Method of Treating Cancer by Inhibition of DNA Repair Proteins

ABSTRACT

Methods of treating cancer using antisense oligonucleotides directed against DNA double-strand break repair proteins such as BRCA2 or RAD51 are provided. The antisense oligonucleotides can he used alone, in tandem or in combination with other cancer therapies, in particular with therapies that lead to DNA damage, inhibition of DNA repair or inhibition of DNA synthesis, such as radiation, platinum drugs, alkylating agents, PARP inhibitors, or inhibitors of thymidylate synthase.

FIELD OF THE INVENTION

The present invention relates to the field of cancer therapy and, inparticular, to the use of antisense oligonucleotides directed againstDNA repair proteins involved in the repair of double strand DNA breaksin the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer is characterized by genetic instability both at the chromosomallevel and at the nucleotide level. The acquisition of certain mutationsconfers a selective advantage to the cancer cells and is critical incancer progression. A diverse array of defects in both DNA polymerasesand DNA repair enzymes appears to contribute to the increased geneticinstability observed in cancer cells (Hanahan et al, 2011, Cell, 144:646-674). Although necessary to confer a selective advantage to cancercells, excessive instability in the cancer cell genome is suggested tobe incompatible with cell viability. Treatment of cancer by increasingthe genetic instability of cancer cells beyond the threshold over whichthe cancer cells are no longer viable has been proposed as analternative therapeutic approach (Loeb, 2011, Nature Reviews Cancer 11,450-457).

A variety of anticancer drugs, including platinum drugs, alkylatingagents, and anthracyclines, share DNA as a common target of biologicalactivity. Covalent binding of drugs to DNA or other interactions thatinterfere with transcription and/or replication initiates a series ofevents that, although intended to rescue the cell for furtherproliferation, may eventually lead to cell death. This depends onvarious factors, including the degree of drug binding, the activity ofthe DNA repair systems, and the balance between pro- and anti-apoptoticmechanisms in the cell. The cytotoxic effect, and therefore the clinicaleffectiveness, of these classes of drugs can potentially be reduced bythe action of DNA repair enzymes and damage-signal molecules. Incontrast, if DNA repair is deficient, a phenotype which may contributeto malignant progression (mutator phenotype), the resulting tumour maybe more susceptible to DNA-damaging agents (1). One deficiency thatcontributes to oncogenesis but leaves the tumour vulnerable to targetedtreatment directed against other genes or gene products capable ofcompensating for the original deficiency is referred to as “syntheticlethality.”

Synthetic lethality (also known as Synthetic Sickness/Lethality or“SSL”) can be described as follows: “Two genes have a SSL relationshipwhen inhibition or mutation of either gene alone does not cause loss ofviability/sickness, but simultaneous inhibition of both genes results inreduced cell viability or an impairment of cellular health/fitness.”(Brough et al, 2011, Curr Op in Gen and Dev., 21: 34-41). Brough et al.also describe how SSL relationship can be used to identify therapeuticoptions in that if one gene in an SSL relationship is a tumoursuppressor gene, then its synthetic lethal partner becomes a candidatetherapeutic target for tumours with a defined tumour suppressor genedysfunction. SSL can occur between genes acting in the same biochemicalpathway or in distinct but compensatory pathways, and components of thesame pathway often share the same SSL partners. Synthetic lethality canbe mimicked by targeted therapies (Chan et al, Nat. Rev. Drug. Discov.,10: 351-364).

Two DNA repair-associated proteins that are known to be deficient inseveral forms of inherited cancer susceptibility are BRCA1 and BRCA2.These proteins are intimately involved with proteins such as PALB2 andRAD51 in mediating homologous recombination (HR)-dependent DNA (HR-DD)repair, the most precise of several repair pathways (8-10). BRCA2mediates binding of RAD51 to short, single-stranded DNA as part of therecognition of DNA strand breaks and initiation of DNA repair (8, 9).BRCA1 is then involved in processing of the free end of a DNA strandbreak, whereas BRCA2 is essential to a strand-exchange step of HR (9).The repair complex includes direct physical interactions between BRCA1,BRCA2, PALB2, BARD1 and RAD51, not only at sites of DNA damage but alsoat chromosomal foci in mitotic cells (8).

Another important protein in mediating base-excision DNA repair (singlestrand DNA break repair) is the enzyme PARP1 (poly[adenosine diphosphate(ADP)-ribose] polymerase) (8). PARP regulates transcription of genesinvolved in other repair mechanisms, including BRCA2 (11). However, PARPis also involved in repair pathways that are independent of BRCA1 andBRCA2 pathways and that tend to be more error-prone (8). If cells aredeficient in BRCA1 or BRCA2, and thus HR-DD repair, the cells becomedependent upon PARP-dependent repair pathways (8). In this case, repairis very sensitive to inhibitors of PARP, and cells tend to accumulatereplication-generated errors that would normally be repairedimmediately, leading eventually to cell cycle arrest and cell death (8).

Clinical examples of drug-hypersensitivity of DNA-repair-deficienttumours have been described. Familial carcinomas of breast, ovary andprostate with a deficiency of BRCA1 or BRCA2 are more sensitive toolaparib(4-[(3-{[4-cyclopropylcarbonyl)piperazin-1-yl]carbonyl}-4-fluorophenyl)methyl]phthalazin-1(2H)-one;also known as AZD2281). Treatment of BRCA1- or BRCA2-deficient tumourswith olaparib has resulted in good clinical responses (2).

As a method to study the function of specific gene products in cellularprocesses, researchers have utilized the ability of nucleic acid that iscomplementary to mRNA to initiate degradation of that mRNA specifically.This phenomenon, which exists in cells as part of a stringent mechanismof control of mRNA levels as well as an antiviral defence, makes use ofnucleic acids that are referred to as “antisense”. Specificdown-regulation of intracellular proteins can be accomplished with theuse of such antisense nucleic acids that bind specifically, based onsequence matching and Watson-Crick base-pairing, to a selected mRNAtarget. By recruitment of intracellular endonucleases, the target mRNAis destroyed, and the protein usually generated from it disappears withnormal turnover. Full-length antisense mRNA expression vectors arecurrently not of potential clinical use. However, shorter antisensenucleic acids have clinical potential, and one format has already beenused in clinical trials. Several different chemistries of antisensemolecules have been used in experimental systems to specificallydown-regulate an mRNA of interest. Oligonucleotides (OLIGOs) consist ofa single-stranded molecule that is introduced into cultured cells usinga cationic liposomal transfecting agent in order to permeate the cellmembrane, although there is some indication that carriers in the bloodare able to facilitate entry of OLIGOs into cells in vivo.

It has been reported that down-regulation of BRCA2 using an antisenseOLIGO targeted against the region of the translational start siteincreased the sensitivity of tumour cells to mitoxantrone in vitro (6).The authors concluded that these effects could be applied to a BRCA2genetic screening method as a predictor of response to a specifictherapeutic approach. It has also been reported that cells treated witha pool of 4 siRNAs targeting BRCA1 or BRCA2 (Dharmacon, Thermo Fisher,Lafayette, Colo., U.S.A. were more sensitive than control siRNA-treatedcounterparts to cytotoxic activity of a PARP inhibitor (7). In thisstudy, the authors concluded that their synthetic lethal siRNA screenwith chemical inhibitors could be used to define new determinants ofsensitivity and potential therapeutic targets. Both of these two studiesfocussed on the use of BRCA2 inhibition in screening methods; neithersuggested that inhibition of BRCA2 may have therapeutic potential.

U.S. Pat. No.5,837,492 describes materials and methods used to isolateand detect a human breast cancer predisposing gene (BRCA2) and describesgenerally polynucleotides comprising all or a part of a BRCA2 locus,including antisense oligonucleotides.

U.S. Patent Publication No. 2004/0097442 describes compounds,compositions and methods for modulating the expression of BRCA2 regiontranscription unit CG005, which is a region of the BRCA2 locus that isoutside the BRCA2 gene itself. The compositions compriseoligonucleotides targeted to nucleic acid encoding BRCA2 regiontranscription unit CG005, i.e. oligonucleotides targeted to mRNAencoding part of the BRCA2 locus other than the BRCA2 gene. Methods ofusing these compounds for the diagnosis and treatment of diseaseassociated with expression of BRCA2 region transcription unit CG005 arealso generally described. United States Patent Publication No.2004/0097442 does not describe antisense oligonucleotides directed tothe mRNA encoding BRCA2.

U.S. Patent Publication No. 2005/0227919 describes methods and meansrelating to the treatment of cancers which are deficient in HR-dependentDNA DSB repair using inhibitors which target base excision repaircomponents such as poly (ADP-ribose) polymerase (PARP).

International Patent Application No. PCT/EP2007/008852 (Publication No.WO 2008/043561) describes pharmaceutical compositions comprisingmodulators of kinases, kinase-binding polypeptides and/or an inhibitorfor influenza virus replication for the prevention and/or treatment ofinfluenza. This application also describes genome-wide screening toidentify human genes that arc relevant for replication of influenzaviruses. Several thousand genes were identified, including BRCA2, andtarget sequences for “knocking down” each gene using siRNA technologywere also identified. Four target sequences within BRCA2 wereidentified.

U.S. Patent Publication No. 2011/0230433 describes methods andcomposition for treatment of cancer by increasing the mutation rate ofcancer cells beyond an error threshold over which the cancer cells areno longer viable.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide methods of treatingcancer by inhibition of DNA repair proteins. In accordance with oneaspect of the present invention, there is provided a method of treatingcancer in a subject comprising administering to the subject an effectiveamount of an antisense oligonucleotide of between 7 and 100 nucleotidesin length comprising a sequence complementary to an mRNA encoding a DNAdouble strand break repair protein.

In accordance with another aspect of the present invention, there isprovided an antisense oligonucleotide of between 7 and 100 nucleotidesin length comprising at least 7 consecutive nucleotides of the sequenceas set forth in any one of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 30, 31, 32,33, 34, 35 or 36.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical composition comprising one or more antisenseoligonucleotides of between 7 and 100 nucleotides in length comprisingat least 7 consecutive nucleotides of the sequence as set forth in anyone of SEQ ID NOs: 2, 3, 14, 15, 30, 31, 32, 33, 34, 35 or 36.

In accordance with another aspect of the present invention, there isprovided a method of treating cancer in a subject comprisingadministering to the subject an effective amount an anti-thymidylatesynthase antisense and an anti-BRCA2 antisense in combination with aplatinum-based chemotherapeutic and a small molecule inhibitor ofthymidylate synthase

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent inthe following detailed description in which reference is made to theappended drawings.

FIG. 1 depicts the inhibitory effect of the anti-BRCA2 antisense OLIGOBR1 on the proliferation of A549b cells.

FIG. 2 depicts the effect of pretreatment with the anti-BRCA2 antisenseOLIGO BR1 on the anti-proliferative activity of olaparib in A549b cells.

FIG. 3 depicts the result of another experiment demonstrating the effectof the anti-BRCA2 antisense OLIGO BR1 on the proliferation of A549bcells.

FIG. 4 depicts the result of another experiment demonstrating the effectof pretreatment with the anti-BRCA2 antisense OLIGO BR1 on theanti-proliferative activity of olaparib in A549b cells.

FIG. 5 depicts the result of a third experiment demonstrating the effectof the anti-BRCA2 antisense OLIGO BR1 on the proliferation of A549bcells.

FIG. 6 depicts the result of a third experiment demonstrating the effectof pretreatment with the anti-BRCA2 antisense OLIGO BR1 on theanti-proliferative activity of olaparib in A549b cells.

FIGS. 7A and 7B depict the effect of pretreatment with the anti-BRCA2antisense OLIGO BR1 or the anti-BRCA2 antisense OLIGO BR3 on theanti-proliferative activity of cisplatin in A549b cells.

FIG. 8 depicts the results of a second experiment showing the effect ofpretreatment with the anti-BRCA2 antisense OLIGO BR3 on theanti-proliferative activity of cisplatin in A549b cells.

FIG. 9 depicts the effect of treating A549b cells with two differentantisense oligonucleotides against BRCA2 (BR1 and BR3) on theproliferation of A549b cells.

FIG. 10 depicts the effect of pretreatment with the anti-BRCA2 antisenseOLIGOs BR1 and BR3 on the anti-proliferative activity of cisplatin inA549b cells.

FIG. 11 depicts the results of a second experiment demonstrating theeffect of pretreatment with the anti-BRCA2 antisense OLIGOs BR1 and BR3on the anti-proliferative activity of cisplatin in A549b cells.

FIG. 12 depicts the effect of treatment with the anti-BRCA2 antisenseOLIGO BR3 and the anti-thymidylate synthase (TS) oligonucleotide OLIGO83 on the proliferation of A549b cells.

FIG. 13 depicts the effect of pretreatment of A549b cells withanti-BRCA2 OLIGO BR1 on the cytotoxicity of melphalan against mediumdensity A549b cells. In this figure and FIGS. 14 to 18, “ODN” was usedin place of “OLIGO” as an abbreviation for oligonucleotide.

FIG. 14 depicts the effect of pretreatment of A549b cells withanti-BRCA2 OLIGO BR1 on the cytotoxicity of carboplatin against mediumdensity A549b cells.

FIG. 15 depicts the effect of pretreatment of A549b cells withanti-BRCA2 OLIGO BR1 on the cytotoxicity of oxaliplatin against lowdensity A549b cells.

FIG. 16 illustrates that antisense TS OLIGO and antisense BR1 OLIGO actindependently to reduce thymidylate synthase and BRCA2 mRNA levels.

FIGS. 17A to 17E illustrate synergistic anti-proliferative effect ofAntisense TS OLIGO and antisense BR1 on A549b cells using concentrationsof oligonucleotides as detailed in the Figure.

FIGS. 18A and 18B illustrate synergistic anti-proliferative effect ofAntisense TS OLIGO and antisense BR3 using concentrations ofoligonucleotides as detailed in the Figure.

FIG. 19 illustrates enhancement of cisplatin cytotoxicity by antisensesiRNA against BRCA2.

FIG. 20 illustrates that four different siRNA molecules against RAD51inhibited proliferation of PANC-1 pancreatic carcinoma cells.

FIG. 21 illustrates that siRNA RADb against RAD51 inhibitedproliferation of A549b cells by over 50% at 2 nM.

FIG. 22 illustrates combined TS siRNA and BRCA2 siRNA enhance A549b cellsensitivity to treatment with cisplatin and SFUdR. All cell numbers areshown as a % of the number of cells treated with control siRNA alone±SD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods of treating cancer usingantisense oligonucleotides targeted against nucleic acids that encodeproteins involved in the repair of double-stranded DNA breaks (DSBs),such as BRCA2, BRCA1, RAD51, PALB2 and DNA-PK.

The antisense oligonucleotides can be used in the treatment of cancer assingle agents (including the use of combinations of the antisenseoligonucleotides) or they may be used in combination with other cancertherapies.

As is known in the art, a number of cancer therapies act by damaging DNAand/or impairing DNA repair or synthesis. Resistance to such therapiesmay arise due to the ability of the cells to repair the DNA damage viavarious DNA repair pathways and/or DNA synthesis. One embodiment of thepresent invention, therefore, provides for the use of the antisenseoligonucleotides in combination with a cancer therapy that damages DNAand/or inhibits DNA repair or synthesis. Without being limited by anyparticular theory, the efficacy of such combinations may be due to thefact that cancer cells in general appear to have an inherently highermutation rate than normal cells and are thus more dependent on DNArepair than normal cells. Many types of cancer cells also have defectsin their mechanisms for repairing DNA damage. As such, cancer cells arelikely to be more vulnerable than normal cells both to DNA damagingagents and to inhibitors of those remaining DNA repair or synthesispathways which are still functional. Accordingly, treatment of cancerpatients with antisense oligonucleotides that target proteins involvedin repairing double-strand DNA breaks can result inhibition of cancercell growth and/or proliferation and also in enhanced cytotoxicity oftherapies that induce DNA damage in cancer cells or that inhibitalternative DNA repair pathways or DNA synthesis pathways. Normal,non-cancerous cells, however, should be able to repair the DNA damageand thus survive treatment. Furthermore, enhancement of the anti-canceractivity of cancer therapies such as DNA-damaging agents could lead tothe use of lower concentrations of the agents to achieve the sameresults, which in turn would decrease common toxicities related to theuse of these agents.

In accordance with one embodiment of the invention, antisenseoligonucleotides targeted to a nucleic acid encoding a DNA DSB repairprotein are used to induce a decrease in expression in the targetedprotein thereby increasing the genetic instability in the cancer cellbeyond a threshold over which the cancer cells are no longer viable.Accordingly, the methods provided by the present invention areapplicable to a wide variety of cancers.

In accordance with one embodiment of the invention, antisenseoligonucleotides targeted to a nucleic acid encoding a DNA DSB repairprotein are used to induce a decrease in expression in the targetedprotein in a patient allowing the patient to obtain greater benefit fromtreatment with a DNA damaging agent and/or an inhibitor of DNA repair orsynthesis. Accordingly, the methods provided by the present inventionare applicable to a wide variety of cancers.

In accordance with one embodiment of the invention, antisenseoligonucleotides targeted to a nucleic acid encoding a DNA DSB repairprotein are used to induce a decrease in expression in the targetedprotein in a patient, thus creating or mimicking a “synthetic lethal”situation and allowing the patient to obtain greater benefit fromtreatment with a DNA damaging agent and/or an inhibitor of DNA repair orsynthesis.

In certain embodiments, the invention encompasses the use of theantisense oligonucleotides targeted to a nucleic acid encoding a DNA DSBrepair protein in the treatment of cancers in which there is already aDNA repair defect. In these embodiments, the antisense oligonucleotidemay target a DNA DSB repair protein in which there is already a partialdefect, or it may target a DNA DSB repair protein belonging to the sameor an alternative DNA repair pathway.

As an example, antisense oligonucleotides targeted to a nucleic acidencoding a BRCA2 protein are capable of inhibiting cancer cell growthand/or proliferation and of potentiating the anti-proliferative effectsof drugs such as the PARP inhibitor, olaparib, and the platinum drug,cisplatin, as well as compounds (such as small molecules or antisenseoligonucleotides) that inhibit thymidylate synthase (TS). Thus, in oneembodiment of the invention, antisense oligonucleotides targeted to anucleic acid encoding a DNA DSB repair protein are used in the treatmentof cancer in combination with cancer therapies that result in DNA damage(such as platinum drugs, alkylating agents, and radiation), or thattarget a range of DNA repair pathways (such as PARP inhibitors). Inanother embodiment, antisense oligonucleotides targeted to a nucleicacid that encodes a DNA DSB repair protein are used in the treatment ofcancer in combination with cancer therapies that impact DNA synthesis,for example anti-cancer agents that inhibit thymidylate synthase (TS).In another embodiment, antisense oligonucleotides targeted to a nucleicacid encoding a DNA DSB repair protein are used as single agents in thetreatment of cancer.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The term “antisense oligonucleotide,” as used herein, refers to anoligonucleotide comprising a sequence that is complementary to the mRNAtranscribed from a target gene. In the context of the present invention,the target gene is the gene encoding a DNA DSB repair protein such as,for example, BRCA2 or RAD51.

The term “anti-proliferative” or “anti-proliferative activity”, as usedherein, means a reduction in total cell number in treated versuscontrol. OLIGOs that have an anti-proliferative activity include thoseOLIGOs that are cytotoxic, induce apoptosis, arrest or delay the cellcycle, alter cell size, or are a combination thereof.

The term “oligonucleotide,” as used herein, means a polymeric form ofnucleotides of at least 7 nucleotides in length comprising eitherribonucleotides or deoxynucleotides or modified forms of either type ofnucleotide. The term includes single and double stranded forms of DNA orRNA. In general, oligonucleotides are between about 7 and about 100nucleotides in length.

“Relative cell density” refers to the relative density of live cells atthe end of an assay.

The term “selectively hybridize” as used herein refers to the ability ofa nucleic acid molecule to bind detectably and specifically to a secondnucleic acid molecule. Oligonucleotides selectively hybridize to targetnucleic acid strands under hybridization and wash conditions thatminimise appreciable amounts of detectable binding to non-specificnucleic acid molecules. High stringency conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein.

Typically, hybridization and washing conditions are performed at highstringency according to conventional hybridization procedures. Washingconditions are typically 1-3×SSC, 0.1-1% SDS, 50-70° C. with a change ofwash solution after about 5-30 minutes.

The term “corresponds to” as used herein with reference to nucleic acidsequences means a polynucleotide sequence that is identical to all or aportion of a reference polynucleotide sequence. In contradistinction,the term “complementary to” is used herein to mean that thepolynucleotide sequence is identical to all or a portion of thecomplement of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

The following terms are used herein to describe the sequencerelationships between two or more polynucleotides: “reference sequence,”“window of comparison,” “sequence identity,” “percent (%) sequenceidentity” and “substantial identity.” A “reference sequence” is adefined sequence used as a basis for a sequence comparison; a referencesequence may be a subset of a larger sequence, for example, as a segmentof a full-length cDNA, mRNA or gene sequence, or may comprise a completecDNA, mRNA or gene sequence. Generally, a reference polynucleotidesequence is at least 20 nucleotides in length, and often at least 50nucleotides in length.

A “window of comparison”, as used herein, refers to a conceptual segmentof the reference sequence of at least 15 contiguous nucleotide positionsover which a candidate sequence may be compared to the referencesequence and wherein the portion of the candidate sequence in the windowof comparison may comprise additions or deletions (i.e. gaps) of 20percent or less as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The present invention contemplates various lengths for thewindow of comparison, up to and including the full length of either thereference or candidate sequence. In one embodiment, the window ofcomparison is the full length of the candidate sequence. Optimalalignment of sequences for aligning a comparison window may be conductedusing the local homology algorithm of Smith and Waterman (Adv. Appl.Math. (1981) 2:482), the homology alignment algorithm of Needleman andWunsch (J. Mol. Biol. (1970) 48:443), the search for similarity methodof Pearson and Lipman (Proc. Natl. Acad. Sci. (U.S.A.) (1988) 85:2444),using computerised implementations of these algorithms (such as GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 573 Science Dr., Madison, Wis.),using publicly available computer software such as ALIGN or Megalign(DNASTAR), or by inspection. The best alignment (i.e. resulting in thehighest percentage of identity over the comparison window) is thenselected.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e. on a nucleotide-by-nucleotide basis) over the window ofcomparison.

The term “percent (%) sequence identity,” as used herein with respect toa reference sequence is defined as the percentage of nucleotide residuesin a candidate sequence that are identical with the residues in thereference polynucleotide sequence over the window of comparison afteroptimal alignment of the sequences and introducing gaps, if necessary,to achieve the maximum percent sequence identity, without consideringany conservative substitutions as part of the sequence identity.

The term “substantial identity” as used herein denotes a characteristicof a polynucleotide sequence, wherein the polynucleotide comprises asequence that has at least 50% sequence identity as compared to areference sequence over the window of comparison. In various embodimentsof the invention, polynucleotide sequences having at least 60% sequenceidentity, at least 70% sequence identity, at least 80% sequenceidentity, at least 90% sequence identity and at least 95% sequenceidentity as compared to a reference sequence over the window ofcomparison are considered to have substantial identity with thereference sequence.

The terms “therapy” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of improving arecipient's status. The improvement can be subjective or objective andis related to the amelioration of the symptoms associated with,preventing the development of, or altering the pathology of a disease,disorder or condition being treated. Thus, the terms therapy andtreatment are used in the broadest sense, and include the prevention(prophylaxis), moderation, reduction, and curing of a disease, disorderor condition at various stages. Prevention of deterioration of arecipient's status is also encompassed by the term. Those in need oftherapy/treatment include those already having the disease, disorder orcondition as well as those prone to, or at risk of developing, thedisease, disorder or condition and those in whom the disease, disorderor condition is to be prevented.

The term “ameliorate” or “amelioration” includes the arrest, prevention,decrease, or improvement in one or more the symptoms, signs, andfeatures of the disease being treated, both temporary and long-term.

The term “subject” or “patient” as used herein refers to a mammal inneed of treatment.

Administration of the compounds of the invention “in combination with”one or more further therapeutic agents, is intended to includesimultaneous (concurrent) administration and consecutive administration.Consecutive administration is intended to encompass administration ofthe therapeutic agent(s) and the compound(s) of the invention to thesubject in various orders and via various routes.

As used herein, the term “about” refers to an approximately +/−10%variation from a given value. It is to be understood that such avariation is always included in any given value provided herein, whetheror not it is specifically referred to.

Target Proteins

Cells comprise distinct pathways for mediating the repair of differenttypes of DNA damage. Such pathways include base excision repair,homologous recombination-dependent DNA double strand break (HR-DD)repair, non-homologous end-joining (NHEJ), nucleotide excision repair,and mismatch repair. HR-DD repair and NHEJ pathways are responsible forthe repair of double strand DNA breaks (DSBs). Antisenseoligonucleotides according to the present invention target nucleic acidsthat encode proteins in the HR-DD or NHEJ pathways, both of which areinvolved in DNA DSB repair.

As used herein, the term “DNA DSB repair protein” refers to a proteininvolved in either the HR-DD pathway or the NHEJ pathway for repairingDNA DS breaks.

In one embodiment, antisense oligonucleotides for use in accordance withthe present invention are designed to target a nucleic acid encoding aDNA DSB repair protein, wherein the DNA DSB protein is involved in theHR-DD repair pathway. Non-limiting examples of key proteins that areinvolved in this pathway include, for example, BRCA1, BRCA2, PALB2 andRAD51. In one embodiment, antisense oligonucleotides for use inaccordance with the present invention are designed to target a nucleicacid encoding the BRCA2 protein or the RAD51 protein.

In one embodiment, antisense oligonucleotides for use in accordance withthe present invention are designed to target a nucleic acid encoding aDNA DSB repair protein, wherein the DNA DSB protein is involved in theNHEJ repair pathway. One of the key proteins in this pathway isDNA-dependent protein kinase (DNA-PK), which includes a catalyticsubunit, DNA-PK_(CS), and a DNA-end binding heterodimer, Ku.

Antisense Oligonucleotides Selection and Characteristics

Antisense oligonucleotides for use in accordance with the presentinvention are designed to target a nucleic acid encoding a DNA DSBrepair protein. The sequences of the genes of various DNA DSB repairproteins involved in the HR-DD or NHEJ repair pathways are known in theart. For example, the sequence of the BRCA2 mRNA is available underGenBank™ Accession No. NM_000059.3 and the sequence of the BRCA2 gene isavailable under GenBank™ Accession No. NG_012772.1. Likewise, thesequences of the RAD51 mRNA (GenBank™ Accession No. NM_001164269.1),RAD51 gene (GenBank™ Accession No. NG_012120.1), BRCA1 mRNA (GenBank™Accession No. NM_007294), BRCA1 gene (GenBank™ Accession No.NG_005905.2), PALB2 mRNA (GenBank™ Accession No. NM_024675.3), PALB2gene, (GenBank™ Accession No. NG_007406.1), DNA-PK mRNA (GenBank™Accession No. NM_001081640.1), and DNA-PK gene (GenBank™ Accession No.NG_023435.1) are also publicly available.

In targeting the antisense oligonucleotides to the selected gene, adetermination is made of a site or sites within this gene or its mRNAfor the antisense interaction to occur such that the desired effect, forexample, modulation of expression of the protein encoded by the geneand/or inhibition of cancer cell growth or proliferation, will result.Once the target site or sites have been identified, oligonucleotides arechosen that are sufficiently complementary (i.e. hybridize withsufficient strength and specificity) to the target to give the desiredresult.

Generally, antisense oligonucleotides can be targeted to the 5′untranslated region (5′-UTR), the translation initiation or start codonregion, the coding sequence (or open reading frame (ORF)), thetranslation termination or stop codon region, or the 3′ untranslatedregion (3′-UTR) of a gene. One embodiment of the present inventionprovides for antisense oligonucleotides targeted to the coding region orthe 3′-UTR of the target mRNA.

The antisense oligonucleotides in accordance with the present inventionare selected such that the antisense sequence exhibits the leastlikelihood of forming duplexes, hairpins or dimers, and contains minimalor no homooligomer/sequence repeats. The oligonucleotide may furthercontain a GC clamp One skilled in the art will appreciate that theseproperties can be determined qualitatively using various computermodelling programs, for example, the program OLIGO® Primer AnalysisSoftware, Version 5.0 (distributed by National Biosciences, Inc.,Plymouth, Minn.).

In order to be effective, conventional antisense oligonucleotides aretypically less than about 100 nucleotides in length, for example,between 7 and 100 nucleotides in length. In one embodiment of thepresent invention, the antisense oligonucleotides are less than about 50nucleotides in length, for example between about 7 and about 50nucleotides in length. In another embodiment, the antisenseoligonucleotides are between about 10 and about 50 nucleotides inlength. In a further embodiment, the antisense oligonucleotides arebetween about 12 and about 50 nucleotides in length. In otherembodiments, the antisense oligonucleotides are less than about 35nucleotides in length, for example between about 7 and about 35nucleotides in length, between about 10 and about 35 nucleotides,between about 12 and about 35 nucleotides, or between about 15 and 35nucleotides. In other embodiments, the antisense oligonucleotides areless than about 30 nucleotides in length, for example between about 15and 30 nucleotides, or between about 12 and 30 nucleotides. In otherembodiments, the antisense oligonucleotides are less than about 25nucleotides in length, for example, between about 15 and 25 nucleotides,and between about 12 and about 25 nucleotides in length.

In one embodiment of the present invention, the antisenseoligonucleotides are complementary to a portion of the mRNA transcribedfrom the BRCA2 gene. In another embodiment of the present invention, theantisense oligonucleotides are complementary to a portion of the codingregion or the 3′-UTR of the BRCA2 mRNA.

Examples of suitable target sequences within the BRCA2 gene or mRNA forthe design of antisense oligonucleotides are known in the art andadditional examples are provided herein. For example, Dharmacon Inc.(Lafayette, Colo.) provides a number of siRNA sequences targeted toBRCA2 gene that could serve as the basis for the design of antisenseoligonucleotides. Examples of antisense oligonucleotide and siRNAsequences known in the art are provided in Table 1 below.

TABLE 1  Antisense Oligonucleotides and siRNA SequencesTargeted to BRCA2 SEQ ID NO Sequence Origin 4 5′-CAGCGTTTGTGTInternational Patent  ATCGGGCA-3′ Application Publication No.WO2008/043561 5 5′-TTGGATCCAATA J. Natl. Cancer Inst., 1998, GGCAT-3′Vol. 90, pp. 978-985 6 5′-TACGTACTCCA International Patent GAACATTTAA-3′ Application Publication No. WO2008/043561 75′-TTGGAGGAATAT International Patent  CGTAGGTAA-3′Application Publication No. WO2008/043561 8 5′-CAGGACACAATTInternational Patent  ACAACTAAA-3′ Application Publication No.WO2008/043561 9 5′-UAAAUAGCAAGU Dharmacon Inc. CCGUUUC-3′ 105′-UAAUGAAGCAUC Dharmacon Inc. UGAUACC-3′ 11 5′-UAUUAAACCUGCDharmacon Inc. AUUCUUC-3′ 12 5′-GUAUCUCUUGAC Dharmacon Inc. GUUCCUUA-3′

In one embodiment, the antisense oligonucleotides for use in accordancewith the present invention comprise a sequence that is complementary toa portion of the BRCA2 mRNA. In one embodiment, the antisenseoligonucleotides for use in accordance with the present inventioncomprise a sequence that is identical or substantially identical to oneof the sequences identified in Table 1 above. In one embodiment, theantisense oligonucleotides comprise a sequence that is complementary toa portion of the coding sequence of the BRCA2 mRNA. In anotherembodiment, the antisense oligonucleotides comprise a sequence that iscomplementary to a portion of the 3′-UTR of the BRCA2 mRNA. In oneembodiment, the antisense oligonucleotide against BRCA2 is other than5′-CAGCGTTTGTGTATCGGGCA-3′ (SEQ ID NO:4). In another embodiment, theantisense oligonucleotide against BRCA2 is other than5′-TTGGATCCAATAGGCAT-3′ (SEQ ID NO:5).

Additional examples of suitable antisense oligonucleotides targeted tothe BRCA2 mRNA include the following (see Example 1 provided herein):

(SEQ ID NO: 1) 5′-GUAUCUUTTGACGTUCCUUA-3′ (SEQ ID NO: 2)5′-UACCAGCGAGCAGGCCGAGU-3′ (SEQ ID NO: 3) 5′-UGCCCGATACACAAACGCUG-3′(SEQ ID NO: 13) 5′-GTATCTCTTGACGTTCCTTA-3′ (SEQ ID NO: 14)5′-TACCAGCGAGCAGGCCGAGT-3′ (SEQ ID NO: 15) 5′-TGCCCGATACACAAACGCTG-3′(SEQ ID NO: 41) 5′-GUAUCUCUUGACGUUCCUUA-3′ (SEQ ID NO: 43)5′-UGCCCGAUACACAAACGCUG-3′

In one embodiment of the present invention, the antisenseoligonucleotide comprises at least 7 consecutive nucleotides of any oneof the sequences set forth in SEQ ID NOs: 1, 2, 3, 13, 14 or 15. Inanother embodiment, the antisense oligonucleotide comprises at least 7and no more than 19 consecutive nucleotides of the antisenseoligonucleotide sequence set forth in SEQ ID NO:4.

In one embodiment of the present invention, the antisenseoligonucleotides comprise a sequence that is complementary to a portionof the mRNA transcribed from the RAD51 gene. In one embodiment, theantisense oligonucleotides comprise a sequence that is complementary toa portion of the coding sequence of the RAD51 mRNA. In anotherembodiment, the antisense oligonucleotides comprise a sequence that iscomplementary to a portion of the 3′-UTR of the RAD51 mRNA. Examples ofsuitable target sequences within the RAD51 mRNA for the design ofantisense oligonucleotides are known in the art and include those shownbelow.

[SEQ ID NO: 26] 5′-CUGCAUCUGCAUUGCCAUUA-3′(Sak et al. 2005, Br j Cancer 92: 1089-1097) [SEQ ID NO: 27]5′-GGCUUCACUAAUUCC-3′ (Raderschall et al. 2002, J Cell Sci 115: 153-164)[SEQ ID NO: 28] 5′-GUAAUGGCAAUGCAGAUGC-3′ (Raderschall et al. ibid.)

An additional example of a suitable antisense oligonucleotide targetedto RAD51 would be an antisense oligonucleotide targeted to all or aportion (for example, at least 7, 8, 9 or 10 consecutive nucleotides) ofthe following target sequence in the 3′-UTR:

[SEQ ID NO: 29] 1734 5′-GAAUGGGUCUGCACAGAUUC-3′

An example of such an antisense oligonucleotide is:

[SEQ ID NO: 44] 5′-GAATCTGTGCAGACCCATTC-3′

In one embodiment of the present invention, the antisenseoligonucleotides comprise a sequence that is complementary to a portionof the mRNA transcribed from the DNA-PK gene. In one embodiment, theantisense oligonucleotides comprise a sequence that is complementary toa portion of the coding sequence of the DNA-PK mRNA. In anotherembodiment, the antisense oligonucleotides comprise a sequence that iscomplementary to a portion of the 3′-UTR of the DNA-PK mRNA. Examples ofantisense oligonucleotide sequences that are targeted to sequenceswithin the DNA-PK mRNA are provided below.

5′-GCAAGCCAGCTGAGGGCACA-3′ [SEQ ID NO:31], which is targeted to part ofthe protein-coding region (positions 874 to 855) of the DNA-PK mRNA.

5′-GGGCATTCCAAGGCTTCCCCA-3′ [SEQ ID NO:32], which is targeted to part ofthe 3′-UTR (positions 12719 to 12699) of the DNA-PK mRNA.

5′-GGGCTCCCATCCTTCCCAGG-3′ [SEQ ID NO:33], which is targeted to part ofthe 3′-UTR (positions 12342 to 12323) of the DNA-PK mRNA.

5′-AGGGGCCTTCTCATGACCCAGG-3′ [SEQ ID NO:34], which is targeted to partof the 3′-UTR (positions 12159 to 12180) of the DNA-PK mRNA.

5′-ACTGCTGGATTGGCACCTGCT-3′ [SEQ ID NO:35], which is targeted to part ofthe 3′-UTR (positions 12117 to 12137) of the DNA-PK mRNA.

5′-TGGGGTCTGTTGCCTGGTCC-3′ [SEQ ID NO:36], which is targeted to part ofthe 3′-UTR (positions 12307 to 12288) of the DNA-PK mRNA.

It is understood in the art that an antisense oligonucleotide need nothave 100% identity with the complement of its target sequence. Theantisense oligonucleotides in accordance with the present invention havea sequence that is at least about 75% identical to the complement oftheir target sequence. In one embodiment of the present invention, theantisense oligonucleotides have a sequence that is at least about 90%identical to the complement of the target sequence. In anotherembodiment, they have a sequence that is at least about 95% identical tothe complement of target sequence, allowing for gaps or mismatches ofseveral bases. In a further embodiment, they are at least about 98%identical to the complement of the target sequence. Identity can bedetermined, for example, by using the BLASTN program of the Universityof Wisconsin Computer Group (GCG) software or provided on the NCBIwebsite.

In one embodiment, the antisense oligonucleotide is capable ofdecreasing or ablating the expression of the DNA DSB repair gene towhich it is targeted. Methods of determining the ability of antisenseoligonucleotides to decrease expression of a target gene are well-knownin the art and may determine the decrease in expression at the nucleicacid level or the protein level or both. For example, after incubationof cells from an appropriate cell line with the antisenseoligonucleotide, the expression of the DNA DSB repair mRNA or proteincan be determined using standard techniques known in the art. Numeroussuch techniques are available to the skilled worker, including DNAarrays, microarrays, protein arrays, proteomics, Northern blots, RT-PCRanalysis, Western blot, and the like.

In the context of this invention, an oligonucleotide (OLIGO) can be anoligomer or polymer of ribonucleic acid (RNA), deoxyribonucleic acid(DNA), or modified RNA or DNA, or combinations thereof. This term,therefore, includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent internucleoside (backbone) linkages aswell as oligonucleotides having non-naturally-occurring portions, whichfunction similarly. Such modified oligonucleotides are often preferredover native forms because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for nucleic acid target andincreased stability in the presence of nucleases. In one embodiment ofthe present invention, the antisense oligonucleotides comprise DNAand/or modified DNA. In another embodiment, the antisenseoligonucleotides comprise RNA and/or modified RNA. In anotherembodiment, the antisense oligonucleotides comprise both DNA and RNA,and/or modified versions thereof.

As is known in the art, a nucleoside is a base-sugar combination and anucleotide is a nucleoside that further includes a phosphate groupcovalently linked to the sugar portion of the nucleoside. In formingoligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound, with thenormal linkage or backbone of RNA and DNA being a 3′ to 5′phosphodiester linkage. Specific non-limiting examples of modifiedoligonucleotides useful in the present invention includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages As defined in this specification,oligonucleotides having modified backbones include both those thatretain a phosphorus atom in the backbone and those that lack aphosphorus atom in the backbone. For the purposes of the presentinvention, and as sometimes referenced in the art, modifiedoligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleotides.

Exemplary antisense oligonucleotides having modified oligonucleotidebackbones include, for example, those with one or more modifiedinternucleoside linkages that are phosphorothioatcs, chiralphosphorothioates, phosphorodithioatcs, phosphotricstcrs,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

In one embodiment of the present invention, the antisenseoligonucleotide is a phosphorothioated oligonucleotide that comprisesone or more phosphorothioate internucleotide linkages. In anotherembodiment, the antisense oligonucleotide comprises phosphorothioateinternucleotide linkages that link the four, five or six 3′-terminalnucleotides of the oligonucleotide. In a further embodiment, theantisense oligonucleotide comprises phosphorothioate internucleotidelinkages that link all the nucleotides of the oligonucleotide.

Exemplary modified oligonucleotide backbones that do not include aphosphorus atom are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. Such backbones include morpholinolinkages (formed in part from the sugar portion of a nucleoside);siloxane backbones; sulfide, sulfoxide and sulphone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl andthioformacetyl backbones; alkene-containing backbones; sulphamatebackbones; methyleneimino and methylenehydrazino backbones; sulphonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

The present invention also contemplates modified oligonucleotides inwhich both the sugar and the internucleoside linkage of the nucleotideunits are replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. Anexample of such a modified oligonucleotide, which has been shown to haveexcellent hybridization properties, is a peptide nucleic acid (PNA)[Nielsen et al., Science, 254:1497-1500 (1991)]. In PNA compounds, thesugar-backbone of an oligonucleotide is replaced with anamide-containing backbone, in particular an aminoethylglycine backbone.The nucicobascs arc retained and arc bound directly or indirectly toaza-nitrogen atoms of the amide portion of the backbone.

The present invention also contemplates oligonucleotides comprising“locked nucleic acids” (LNAs), which are conformationally restrictedoligonucleotide analogues containing a methylene bridge that connectsthe 2′-O of ribose with the 4′-C (see, Singh et al., Chem. Commun.,1998, 4:455-456). LNA and LNA analogues display very high duplex thermalstabilities with complementary DNA and RNA, stability towards3′-exonuclease degradation, and good solubility properties. Synthesis ofthe LNA analogues of adenine, cytosine, guanine, 5-methylcytosine,thymine and uracil, their oligomerization, and nucleic acid recognitionproperties have been described (see Koshkin et al., Tetrahedron, 1998,54:3607-3630). Studies of mis-matched sequences show that LNA obey theWatson-Crick base pairing rules with generally improved selectivitycompared to the corresponding unmodified reference strands.

Antisense oligonucleotides containing LNAs have been demonstrated to beefficacious and non-toxic (Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97:5633-5638). In addition, the LNA/DNA copolymers werenot degraded readily in blood serum and cell extracts.

LNAs form duplexes with complementary DNA or RNA or with complementaryLNA, with high thermal affinities. The universality of LNA-mediatedhybridization has been emphasized by the formation of exceedingly stableLNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998,120:13252-13253). LNA:LNA hybridization was shown to be the mostthermally stable nucleic acid type duplex system, and the RNA-mimickingcharacter of LNA was established at the duplex level. Introduction ofthree LNA monomers (T or A) resulted in significantly increased meltingpoints toward DNA complements.

Synthesis of 2′-amino-LNA (Singh et al., J. Org. Chem., 1998, 63,10035-10039) and 2′-methylamino-LNA has been described and thermalstability of their duplexes with complementary RNA and DNA strandsreported. Preparation of phosphorothioate-LNA and 2′-thio-LNA have alsobeen described (Kumar et al., Bioorg. Med. Chem. Lett., 1998,8:2219-2222).

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, oligonucleotides may comprise sugars with one ofthe following substituents at the 2′ position: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Examplesof such groups are: O[(CH₂)_(n)O]_(m) CH₃, O(CH₂)_(n) OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂, and O(CH₂)_(n) ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Alternatively, theoligonucleotides may comprise one of the following substituents at the2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties.Specific examples include 2′-methoxyethoxy (2′-O—CH₂ CH₂ OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) [Martin et al., Helv. Chim.Acta, 78:486-504(1995)], 2′-dimethylaminooxyethoxy (O(CH₂)₂ ON(CH₃)₂group, also known as 2′-DMAOE), 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂ CH₂ CH₂ NH₂) and 2′-fluoro (2′-F).

In one embodiment of the present invention, the antisenseoligonucleotide comprises at least one nucleotide comprising asubstituted sugar moiety. In another embodiment, the antisenseoligonucleotide comprises at least one 2′-O-(2-methoxyethyl) or 2′-MOEmodified nucleotide. In another embodiment, the antisenseoligonucleotide comprises at least one 2′-O-methyl or 2′-MOEribonucleotide.

Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include modifications to the nucleobase. Asused herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808; The Concise Encyclopedia Of Polymer Science AndEngineering, (1990) pp 858-859, Kroschwitz, J. I., ed. John Wiley &Sons; Englisch et al., Angewandte Chemie, Int. Ed., 30:613 (1991); andSanghvi, Y. S., (1993) Antisense Research and Applications, pp 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. [Sanghvi, Y.S., (1993) Antisense Research and Applications, pp 276-278, Crooke, S.T. and Lebleu, B., ed., CRC Press, Boca Raton].

Another oligonucleotide modification included in the present inventionis the chemical linkage to the oligonucleotide of one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include, but arenot limited to, lipid moieties such as a cholesterol moiety [Letsingeret al., Proc. Natl. Acad. Sci. USA, 86:6553-6556 (1989)], cholic acid[Manoharan et al., Bioorg. Med. Chem. Let., 4:1053-1060 (1994)], athioether, e.g. hexyl-S-tritylthiol [Manoharan et al., Ann. N.Y. Acad.Sci., 660:306-309 (1992); Manoharan et al., Bioorg. Med. Chem. Lett.,3:2765-2770 (1993)], a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20:533-538 (1992)], an aliphatic chain, e.g. dodecandiol orundecyl residues [Saison-Behmoaras et al., EMBO J., 10:1111-1118 (1991);Kabanov et al., FEBS Lett., 259:327-330 (1990); Svinarchuk et al.,Biochimie, 75:49-54 (1993)], a phospholipid, e.g.di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36:3651-3654 (1995); Shea et al., Nucl. Acids Res.,18:3777-3783 (1990)], a polyamine or a polyethylene glycol chain[Manoharan et al., Nucleosides & Nucleotides, 14:969-973 (1995)], oradamantane acetic acid [Manoharan et al., Tetrahedron Lett.,36:3651-3654 (1995)], a palmityl moiety [Mishra et al., Biochim.Biophys. Acta, 1264:229-237 (1995)], or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety [Crooke et al., J. Pharmacol.Exp. Ther., 277:923-937 (1996)].

One skilled in the art will recognise that it is not necessary for allpositions in a given oligonucleotide to be uniformly modified. Thepresent invention, therefore, contemplates the incorporation of morethan one of the aforementioned modifications into a singleoligonucleotide or even at a single nucleoside within theoligonucleotide.

In one embodiment of the present invention, the antisenseoligonucleotides are gapmers. As used herein, the term “gapmer” refersto an antisense oligonucleotide comprising a central region (a “gap”)and a region on either side of the central region (the “wings”), whereinthe gap comprises at least one modification difference compared to eachwing. Such modifications include nucleotide, internucleoside linkage,and sugar modifications as well as the absence of modification(unmodified RNA or DNA). Thus, in certain embodiments, the nucleotidelinkages in each of the wings are different from the nucleotide linkagesin the gap. In certain embodiments, each wing comprises modifiednucleotides and the gap comprises nucleotides that do not comprise thatmodification. In certain embodiments the nucleotides in the gap and thenucleotides in the wings all comprise modified nucleotides, but themodifications in the gap are different from the modifications in each ofthe wings. In certain embodiments, the modifications in the wings arethe same as one another. In certain embodiments, the modifications inthe wings are different from each other. In certain embodiments,nucleotides in the gap are unmodified and nucleotides in the wings aremodified. In certain embodiments, the modification(s) within each wingare the same. In certain embodiments, the modification(s) in one wingare different from the modification(s) in the other wing. In certainembodiments, the nucleotide linkages are the same in the gap and in thewings, but the wings comprise modified nucleotides whereas the gap doesnot. In one embodiment, the nucleotides in the wings comprise 2′-MOEmodifications and the nucleotides in the gap do not.

In the context of the present invention, an antisense oligonucleotide is“nuclease resistant” when it has either been modified such that it isnot susceptible to degradation by DNA and RNA nucleases or alternativelyhas been placed in a delivery vehicle which in itself protects theoligonucleotide from DNA or RNA nucleases. Nuclease-resistantoligonucleotides include, for example, methyl phosphonates,phosphorothioates, phosphorodithioates, phosphotriesters, and morpholinooligomers. Suitable delivery vehicles for conferring nuclease resistanceinclude, for example, liposomes. In one embodiment of the presentinvention, the antisense oligonucleotides are nuclease-resistant.

In some embodiments of the present invention, the antisense sequencesmay be provided in the context of RNAi constructs comprising sequencesspecific for proteins involved in the repair of double-stranded DNAbreaks (DSBs), such as BRCA2, BRCA1, RAD51, PALB2 and DNA-PK.

In one embodiment of the present invention, the RNAi construct comprisesa single-stranded polynucleotide that forms a hairpin structure whichincludes a double-stranded stem and a single-stranded loop, wherein thedouble-stranded stem can be cleaved by Dicer to produce an siRNA.

In one embodiment, the RNAi construct comprises a double-stranded(dsRNA) construct. The RNAi constructs may be modified to increasestability or increase cellular uptake.

The present invention further contemplates antisense oligonucleotidesthat contain groups for improving the pharmacokinetic properties of theoligonucleotide, or groups for improving the pharmacodynamic propertiesof the oligonucleotide.

In embodiments of the present invention where antisense oligonucleotidesdirected to nucleic acids encoding two or more target proteins are used,each oligonucleotide may be independently modified.

Preparation of the Antisense Oligonucleotides

The antisense oligonucleotides in accordance with the present inventioncan be prepared by conventional techniques well-known to those skilledin the art. For example, the oligonucleotides can be prepared usingsolid-phase synthesis using commercially available equipment, such asthe equipment available from Applied Biosystems Canada Inc.,Mississauga, Canada. As is well-known in the art, modifiedoligonucleotides, such as phosphorothioates and alkylated derivatives,can also be readily prepared by similar methods.

Alternatively, the antisense oligonucleotides can be prepared byenzymatic digestion of the naturally occurring DNA DSB repair proteingene by methods known in the art.

Antisense oligonucleotides can also be prepared through the use ofrecombinant methods in which expression vectors comprising nucleic acidsequences that encode the antisense oligonucleotides are expressed in asuitable host cell. Such expression vectors can be readily constructedusing procedures known in the art. Examples of suitable vectors include,but are not limited to, plasmids, phagemids, cosmids, bacteriophages,baculoviruses and retroviruses, and DNA viruses. One skilled in the artwill understand that selection of the appropriate host cell forexpression of the antisense oligonucleotide will be dependent upon thevector chosen. Examples of host cells include, but are not limited to,bacterial, yeast, insect, plant and mammalian cells.

One skilled in the art will also understand that the expression vectormay further include one or more regulatory elements, such astranscriptional elements, required for efficient transcription of theantisense oligonucleotide sequences. Examples of regulatory elementsthat can be incorporated into the vector include, but are not limitedto, promoters, enhancers, terminators, and polyadenylation signals. Oneskilled in the art will appreciate that selection of suitable regulatoryelements is dependent on the host cell chosen for expression of theantisense oligonucleotide and that such regulatory elements may bederived from a variety of sources, including bacterial, fungal, viral,mammalian or insect genes.

The expression vectors can be introduced into a suitable host cell ortissue by one of a variety of methods known in the art. Such methods canbe found generally described in Sambrook et al., 1992; Ausubel et al.,1989; Chang et al., 1995; Vega et al., 1995; and Vectors: A Survey ofMolecular Cloning Vectors and Their Uses (1988) and include, forexample, stable or transient transfection, lipofection, electroporation,and infection with recombinant viral vectors.

Efficacy of the Antisense Oligonucleotides

The antisense oligonucleotides in accordance with the present inventioncan be tested for their ability to inhibit the growth and/orproliferation of cancer cells in vitro and/or in vivo using standardtechniques. The antisense oligonucleotides can be tested individually,or two or more antisense oligonucleotides can be tested in combination.The antisense oligonucleotides can also be tested in combination withother cancer therapies. Exemplary testing methods are described belowand in the Examples provided herein.

1. In Vitro Testing

Initial determinations of the ability of the antisense oligonucleotidesto attenuate the growth or proliferation of neoplastic cells may be madeusing in vitro techniques if required.

For example, the cytotoxicity of the antisense oligonucleotides can beassayed in vitro using a suitable cancer cell line. In general, cells ofthe selected test cell line are grown to an appropriate density and thetest compound(s) are added. After an appropriate incubation time (forexample, about 48 to 96 hours), cell survival is assessed. Methods ofdetermining cell survival are well known in the art and include, but arenot limited to, the resazurin reduction test (see Fields & Lancaster(1993) Am. Biotechnol. Lab. 11:48-50; O'Brien et al., (2000) Eur.Biochem. 267:5421-5426 and U.S. Pat. No. 5,501,959), the sulforhodamineassay (Rubinstein et al., (1990) J. Natl. Cancer Inst. 82:113-118) orthe neutral red dye test (Kitano et al., (1991) Euro. J. Clin. Investg.21:53-58; West et al., (1992) J. Investigative Derm. 99:95-100).Cytotoxicity is determined by comparison of cell survival in the treatedculture with cell survival in one or more control cultures, for example,untreated cultures, cultures pre-treated with a control compound(typically a known therapeutic) and/or cultures treated individuallywith the components of the antisense oligonucleotide.

Alternatively, the ability of the antisense oligonucleotides to inhibitproliferation of neoplastic cells can be assessed by culturing cells ofa cancer cell line of interest in a suitable medium. After anappropriate incubation time, the cells can be treated with the antisenseoligonucleotide and incubated for a further period of time. Cells arethen counted using a technique known in the art, such as an electronicparticle counter or a haemocytometer, and compared to an appropriatecontrol, as described above.

The antisense oligonucleotides can also be tested in vitro bydetermining their ability to inhibit anchorage-independent growth oftumour cells. Anchorage-independent growth is known in the art to be agood indicator of tumourigenicity. In general, anchorage-independentgrowth is assessed by plating cells from an appropriate cancer cell lineonto soft agar and determining the number of colonies formed after anappropriate incubation period. Growth of cells treated with theantisense oligonucleotides can then be compared with that of cellstreated with an appropriate control (as described above) and with thatof untreated cells.

A variety of cancer cell lines suitable for testing the antisenseoligonucleotides are known in the art and many are commerciallyavailable (for example, from the American Type Culture Collection,Manassas, Va.). In one embodiment of the present invention, in vitrotesting of the antisense oligonucleotides is conducted in a human cancercell line. Examples of suitable cancer cell lines for in vitro testinginclude, but are not limited to, breast cancer cell lines MDA-MB-231 andMCF-7, renal carcinoma cell line A-498, mesothelial cell linesMSTO-211II, NCI-II2052 and NCI-II28, ovarian cancer cell lines OV90 andSK-OV-3, colon cancer cell lines CaCo, HCT116 and HT29, cervical cancercell line HeLa, non-small cell lung carcinoma cell lines A549, A549b,and H1299, pancreatic cancer cell lines MIA-PaCa-2 and AsPC-1, prostaticcancer-cell line PC-3, bladder cancer cell line T24, liver cancer cellline HepG2, brain cancer cell line U-87 MG, melanoma cell line A2058,and lung cancer cell line NCI-H460. Other examples of suitable celllines are known in the art.

If necessary, the toxicity of the antisense oligonucleotides can also beinitially assessed in vitro using standard techniques. For example,human primary fibroblasts can be treated in vitro with theoligonucleotide in the presence of a commercial lipid carrier such asLipofectamine 2000 (LFA2K) (available from Life Technologies,Burlington, Ontario, Canada). Cells are then tested at different timepoints following treatment for their viability using a standardviability assay, such as the trypan-blue exclusion assay. Cells are alsoassayed for their ability to synthesize DNA, for example, using athymidine incorporation assay, and for changes in cell cycle dynamics,for example, using a standard fluorescence-dependent flow cytometricassay.

2. In Vivo Testing

The ability of the antisense oligonucleotides to inhibit tumour growthor proliferation in vivo can be determined in an appropriate animalmodel using standard techniques known in the art (see, for example,Enna, et al., Current Protocols in Pharmacology, J. Wiley & Sons, Inc.,New York, N.Y.).

In general, current animal models for screening anti-tumour compoundsare xenograft models, in which a human or mammalian tumour has beenimplanted into an animal Examples of xenograft models of human cancerinclude, but are not limited to, human solid tumour xenografts in mice,implanted by sub-cutaneous injection and used in tumour growth assays;human solid tumour isografts in mice, implanted by fat pad injection andused in tumour growth assays; human solid tumour orthotopic xenografts,implanted directly into the relevant tissue and used in tumour growthassays; experimental models of lymphoma and leukaemia in mice, used insurvival assays, and experimental models of metastasis in mice.

For example, the antisense oligonucleotides can be tested in vivo onsolid tumours using mice that are subcutaneously grafted bilaterallywith a pre-determined amount of a tumour fragment on day 0. The animalsbearing tumours are mixed before being subjected to the varioustreatments and controls. In the case of treatment of advanced tumours,tumours are allowed to develop to the desired size, animals havinginsufficiently developed tumours being eliminated. The selected animalsare distributed at random into groups that will undergo the treatmentsor act as controls. Animals not bearing tumours may also be subjected tothe same treatments as the tumour-bearing animals in order to be able todissociate the toxic effect from the specific effect on the tumour.Treatment generally begins from 3 to 22 days after grafting, dependingon the type of tumour, and the animals are observed every day. Theantisense oligonucleotides of the present invention can be administeredto the animals, for example, by bolus infusion or intraperitonealinjection (Ferguson et al., 2007, Eur J Cancer Supplements, Abstract B153, p. 211-212). The different animal groups are weighed about 3 or 4times a week until the maximum weight loss is attained, after which thegroups are weighed less frequently, for example, at least once a weekuntil the end of the trial.

The tumours are measured about 2 or 3 times a week until the tumourreaches a pre-determined size and/or weight, or until the animal dies ifthis occurs before the tumour reaches the pre-determined size/weight.The animals are then sacrificed and the tissue histology, size and/orproliferation of the tumour assessed.

For the study of the effect of the compositions on leukaemias, theanimals are grafted with a particular number of cells, and theanti-tumour activity is determined by the increase in the survival timeof the treated mice relative to the controls.

To study the effect of the antisense oligonucleotides of the presentinvention on tumour metastasis, tumour cells are typically treated withthe composition ex vivo and then injected into a suitable test animal.The spread of the tumour cells from the site of injection is thenmonitored over a suitable period of time by standard techniques. Inanother technique, test animals in which a primary tumour has beenestablished can be used. The primary tumour is removed when it reaches acertain size and/or after it has been treated with a certain protocol,and the appearance of metastases is monitored. Alternatively, afterremoval of the primary tumour, the animal can be treated to determinewhether growth of metastases can be inhibited in comparison tono-treatment control animals.

In vivo toxic effects of the oligonucleotides can be evaluated bymeasuring their effect on animal body weight during treatment and byperforming haematological profiles and liver enzyme analysis after theanimal has been sacrificed.

TABLE 2 Examples of Xenograft Models of Human Cancer Cancer Model CellType Tumour Growth Assay Prostate (PC-3, DU145) Human solid tumourxenografts Breast (MDA-MB-231, MVB-9) in mice (sub-cutaneous Colon(HT-29) injection) Lung (NCI-H460, NCI-H209, A549) Pancreatic (ASPC-1,SU86.86) Pancreatic: drug resistant (BxPC-3) Skin (A2058, C8161)Cervical (SIHA, HeLa-S3) Cervical: drug resistant (HeLa S3-HU-resistance) Liver (HepG2) Brain (U87-MG) Renal (Caki-1, A498) Ovary(SK-OV-3) Tumour Growth Assay Breast: drug resistant (MDA-CDDP-S4,MDA-MB435-To.1) Human solid tumour isografts in mice (fat pad injection)Survival Assay Human: Burkitts lymphoma (Non- Experimental model ofHodgkin's) (Raji) lymphoma and leukaemia Murine: erythroleukemia (CB7Friend in mice retrovirus-induced), L1210, P388, S49 Experimental modelof lung Human: melanoma (C8161) metastasis in mice Murine: fibrosarcoma(R3)

3. Combination Therapies

As noted above, the antisense oligonucleotides can be tested incombination with another cancer therapy. Combinations comprising two ormore antisense oligonucleotides, or comprising the antisenseoligonucleotide together with another cancer therapy may be moreeffective than each of the components when used alone. Improved efficacycan be manifested, for example, as a less-than-additive effect, whereinthe effect of the combination is greater than the effect of eachcomponent alone, but less than the sum of the effects of the components,or it may be an additive effect, wherein the effect of the combinationis equivalent to the sum of the effects of the components when usedindividually, or it may be a greater-than-additive effect, wherein theeffect of the combination is greater than the sum of the effects of eachcomponent used alone. Greater-than-additive effects may also bedescribed as synergistic. The improved efficacy of the combinations canbe determined by a number of methods known in the art.

For example, such improved efficacy can result in one or more of: (i) anincrease in the ability of the combination to inhibit the growth orproliferation of neoplastic cells when compared to the effect of eachcomponent alone; (ii) a decrease in the dose of one or more of thecomponents being required to bring about a certain effect (i.e. adecrease in the median effective dose or ED₅₀); (iii) decreased toxicityphenomena associated with one or more of the components (i.e. anincrease in the median lethal dose or LD₅₀), and (iv) an improvedtherapeutic index or clinical therapeutic index of the combination whencompared to the therapeutic index/clinical therapeutic index of eachcomponent alone.

As used herein, the term “therapeutic index” is defined as LD₅₀/ED₅₀,where “ED₅₀” is the amount of a compound that produces 50% of themaximum response or effect associated with the compound, or the amountthat produces a pre-determined response or effect in 50% of a testpopulation, and “LD₅₀” is the amount of a compound that has a lethaleffect in 50% of a test population. Thus, a compound with a hightherapeutic index can typically be administered with greater safety thanone with a low therapeutic index. The LD₅₀ is determined in preclinicaltrials, whereas the ED₅₀ can be determined in preclinical or clinicaltrials. Preclinical trials are conducted using an appropriate animalmodel, such as those described herein. The therapeutic index can also bedetermined based on doses that produce a therapeutic effect and dosesthat produce a toxic effect (for example, the ED₉₀ and LD₁₀,respectively).

“Clinical therapeutic index” differs from therapeutic index in that someindices of relative safety or relative effectiveness in patients in aclinical setting cannot be defined explicitly and uniquely. Acombination is considered to demonstrate an improved ClinicalTherapeutic Index, therefore, when it meets one of the followingcriteria as defined by the Food and Drug Administration: demonstratesincreased safety (or patient acceptance) at an accepted level ofefficacy within the recommended dosage range, or demonstrates increasedefficacy at equivalent levels of safety (or patient acceptance) withinthe recommended dosage range, as compared to each of the components inthe combination. Alternatively, during clinical studies, the dose or theconcentration (for example, in solution, blood, serum, plasma) of a drugrequired to produce toxic effects can be compared to the concentrationrequired to achieve the desired therapeutic effects in the population inorder to evaluate the clinical therapeutic index. Methods of clinicalstudies to evaluate the clinical therapeutic index are well known toworkers skilled in the art.

Combinations may also exhibit therapeutic synergy, wherein “therapeuticsynergy” is demonstrated when a combination is therapeutically superiorto one of the components of the combination when used at thatcomponent's optimum dose [as defined in T. H. Corbett et al., (1982)Cancer Treatment Reports, 66:1187]. To demonstrate the efficacy of acombination, it may be necessary to compare the maximum tolerated doseof the combination with the maximum tolerated dose of each of theseparate components in the study in question. This efficacy may bequantified using techniques and equations commonly known to workersskilled in the art [see, for example, T. H. Corbett et al., (1977)Cancer, 40, 2660.2680; F. M. Schabel et al., (1979) Cancer DrugDevelopment, Part B, Methods in Cancer Research, 17:3-51, New York,Academic Press Inc.].

One embodiment of the present invention provides for the use of acombination of two or more antisense oligonucleotides targeted to anucleic acid encoding a DNA DSB repair protein, wherein the effect ofthe combination is greater-than-additive or synergistic. Anotherembodiment of the present invention provides for the use of acombination of an antisense oligonucleotide targeted to a nucleic acidencoding a DNA DSB repair protein and a cancer therapy that damages DNA,inhibits a DNA repair pathway or impacts DNA synthesis, wherein theeffect of the combination is greater-than-additive or synergistic.Another embodiment provides for the use of a combination of one or moreantisense oligonucleotides targeted to a specific DNA DSB repair proteinmRNA with another cancer therapy, such as radiation or achemotherapeutic drug, wherein the effect of the combination isgreater-than-additive or synergistic.

Pharmaceutical Compositions

The antisense oligonucleotide(s) may be administered as a pharmaceuticalcomposition in which the antisense oligonucleotide(s) are admixed withan appropriate pharmaceutically acceptable carrier, diluent, excipientor vehicle.

The pharmaceutical compositions of the present invention may beadministered orally, topically, parenterally, by inhalation or spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques.

The present invention also provides for pharmaceutical compositionscomprising an antisense oligonucleotide associated with a liposomal-typevehicle, such as an artificial membrane vesicle (including a liposome,lipid micelle and the like), microparticle or microcapsule.

The pharmaceutical compositions may be in a form suitable for oral use,for example, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use may be preparedaccording to methods known to the art for the manufacture ofpharmaceutical compositions and may contain one or more agents selectedfrom the group of sweetening agents, flavouring agents, colouring agentsand preserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with suitable non-toxic pharmaceutically acceptable excipientsincluding, for example, inert diluents, such as calcium carbonate,sodium carbonate, lactose, calcium phosphate or sodium phosphate;granulating and disintegrating agents, such as corn starch, or alginicacid; binding agents, such as starch, gelatine or acacia, andlubricating agents, such as magnesium stearate, stearic acid or talc.The tablets can be uncoated, or they may be coated by known techniquesin order to delay disintegration and absorption in the gastrointestinaltract and thereby provide a sustained action over a longer period. Forexample, a time delay material such as glyceryl monosterate or glyceryldistearate may be employed.

Pharmaceutical compositions for oral use may also be presented as hardgelatine capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatine capsules wherein the active ingredient ismixed with water or an oil medium such as peanut oil, liquid paraffin orolive oil.

Aqueous suspensions contain the active compound in admixture withsuitable excipients including, for example, suspending agents, such assodium carboxymethylcellulose, methyl cellulose,hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia; dispersing or wetting agents such as anaturally-occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for example,polyoxyethyene stearate, or condensation products of ethylene oxide withlong chain aliphatic alcohols, for example,hepta-decaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol for example,polyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxy-benzoate, one or more colouring agents, oneor more flavouring agents or one or more sweetening agents, such assucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example, beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and/or flavouring agents may be added to provide palatable oralpreparations. These compositions can be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil, forexample, olive oil or arachis oil, or a mineral oil, for example, liquidparaffin, or it may be a mixture of these oils. Suitable emulsifyingagents may be naturally-occurring gums, for example, gum acacia or gumtragacanth; naturally-occurring phosphatides, for example, soy bean,lecithin; or esters or partial esters derived from fatty acids andhexitol, anhydrides, for example, sorbitan monoleate, and condensationproducts of the said partial esters with ethylene oxide, for example,polyoxyethylene sorbitan monoleate. The emulsions may also containsweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, and/orflavouring and colouring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using suitable dispersing or wettingagents and suspending agents such as those mentioned above. The sterileinjectable preparation may also be sterile injectable solution orsuspension in a non-toxic parentally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Acceptable vehicles andsolvents that may be employed include, but are not limited to, water,Ringer's solution, lactated Ringer's solution and isotonic sodiumchloride solution. Other examples are, sterile, fixed oils which areconventionally employed as a solvent or suspending medium, and a varietyof bland fixed oils including, for example, synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

In one embodiment of the present invention, the pharmaceuticalcomposition comprising the antisense oligonucleotide is formulated forinjection or infusion.

Other pharmaceutical compositions and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy,” Gennaro,A., Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000) (formerly“Remingtons Pharmaceutical Sciences”).

Use of the Antisense Oligonucleotides

The present invention provides for the use of the antisenseoligonucleotides in the treatment of cancer. The antisenseoligonucleotides may be used alone as single agents or may be used incombination with another cancer therapy. When used as a single agent,the antisense oligonucleotides may be used singly or in tandem (i.e. twoantisense oligonucleotides targeting the same DNA DSB repair proteingene or mRNA), or the antisense oligonucleotides may be combined invarious other ways (for example, three or more antisenseoligonucleotides targeting the same DNA DSB repair protein gene or mRNA,or two or more two antisense oligonucleotides each targeting a differentDNA DSB repair protein gene or mRNA).

One embodiment of the present invention provides for the use of one ormore antisense oligonucleotides targeting a specific DNA DSB repairprotein mRNA in the treatment of cancer. Another embodiment provides forthe use of a combination of one or more antisense oligonucleotidestargeting a specific DNA DSB repair protein mRNA and one or moreantisense oligonucleotides targeting a different specific DNA DSB repairprotein mRNA in order to treat cancer. Another embodiment provides forthe use of a combination of one or more antisense oligonucleotidestargeting a specific DNA DSB repair protein mRNA with another cancertherapy, such as radiation or a chemotherapeutic drug.

One embodiment of the invention provides for the use of a combination ofone or more antisense oligonucleotides targeted to the mRNA of a DNA DSBrepair protein mRNA in the HR-DD pathway with one or more antisenseoligonucleotides targeted to the mRNA of a DNA DSB repair protein mRNAin the NHEJ pathway. The use in accordance with this embodiment includesthe use of the antisense oligonucleotides alone or in conjunction withone or more other cancer therapies, such as radiation or achemotherapeutic drug.

The present invention contemplates the use of the antisenseoligonucleotides in the treatment of a variety of cancers. Treatment ofcancer encompasses the use of the antisense oligonucleotides to treat,stabilize or prevent cancer. In this context, treatment with theantisense oligonucleotides may result in, for example, a reduction inthe size of a tumour, the slowing or prevention of an increase in thesize of a tumour, an increase in the disease-free survival time betweenthe disappearance or removal of a tumour and its reappearance,prevention of an initial or subsequent occurrence of a tumour (e.g.metastasis), an increase in the time to progression, reduction of one ormore adverse symptom associated with a tumour, a slowing of tumourregression, or an increase in the overall survival time of a subjecthaving cancer.

Examples of cancers which may be may be treated or stabilized inaccordance with the present invention include, but are not limited tohaematologic neoplasms, including leukaemias and lymphomas; carcinomas,including adenocarcinomas; melanomas and sarcomas. Carcinomas,adenocarcinomas and sarcomas are also frequently referred to as “solidtumours.” Examples of commonly occurring solid tumours include, but arenot limited to, cancer of the brain, breast, cervix, colon, rectum, headand neck, kidney, lung including both small cell and non-small cell lungcancer, ovary, pancreas, prostate, stomach and uterus. Various forms oflymphoma also may result in the formation of a solid tumour and,therefore, in certain contexts may also be considered to be solidtumours.

The term “leukaemia” refers broadly to progressive, malignant diseasesof the blood-forming organs. Leukaemia is typically characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow but can also refer to malignantdiseases of other blood cells such as erythroleukaemia, which affectsimmature red blood cells. Leukaemia is generally clinically classifiedon the basis of (1) the duration and character of the disease—acute orchronic; (2) the type of cell involved—myeloid (myelogenous), lymphoid(lymphogenous) or monocytic, and (3) the increase or non-increase in thenumber of abnormal cells in the blood—leukaemic or aleukaemic(subleukaemic). Leukaemia includes, for example, acute nonlymphocyticleukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia,chronic granulocytic leukaemia, acute promyelocytic leukaemia, adultT-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia,basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronicmyelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilicleukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblasticleukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cellleukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphaticleukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenousleukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cellleukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia,monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia,myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegelileukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocyticleukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cellleukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia.

The term “lymphoma” generally refers to a malignant neoplasm of thelymphatic system, including cancer of the lymphatic system. The two maintypes of lymphoma arc Hodgkin's disease (HD or HL) and non-Hodgkin'slymphoma (NHL). Abnormal cells appear as congregations which enlarge thelymph nodes, form solid tumours in the body, or more rarely, likeleukemia, circulate in the blood. Hodgkins' disease lymphomas include:nodular lymphocyte predominance Hodgkin's lymphoma; classical Hodgkin'slymphoma; nodular sclerosis Hodgkin's lymphoma; lymphocyte-richclassical Hodgkin's lymphoma; mixed cellularity Hodgkin's lymphoma;lymphocyte depletion Hodgkin's lymphoma. Non-Hodgkin's lymphomas includesmall lymphocytic NHL; follicular NHL; mantle cell NHL;mucosa-associated lymphoid tissue (MALT) NHL; diffuse large cell B-cellNHL; mediastinal large B-cell NHL; precursor T lymphoblastic NHL;cutaneous T-cell NHL; T-cell and natural killer cell NHL; mature(peripheral) T-cell NHL; Burkitt's lymphoma; mycosis fungoides; SézarySyndrome; precursor B-lymphoblastic lymphoma; B-cell small lymphocyticlymphoma; lymphoplasmacytic lymphoma; splenic marginal zone B-celllymphoma; nodal marginal zone lymphoma; plasma cellmyeloma/plasmacytoma; intravascular large B-cell NHL; primary effusionlymphoma; blastic natural killer cell lymphoma; enteropathy-type T-celllymphoma; hepatosplenic gamma-delta T-cell lymphoma; subcutaneouspanniculitis-like T-cell lymphoma; angioimmunoblastic T-cell lymphoma;and primary systemic anaplastic large T/null cell lymphoma.

The term “sarcoma” generally refers to a tumour which originates inconnective tissue, such as muscle, bone, cartilage or fat, and is madeup of a substance like embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include soft tissue sarcomas, chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,choriocarcinoma, embryonal sarcoma, Wilms tumour sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented haemorrhagic sarcoma, immunoblasticsarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumour arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma, sublingual melanoma, and superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangioccllular carcinoma, chorionic carcinoma, colorectal carcinoma,colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriformcarcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindricalcarcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum,embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma,carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinouscarcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandularcarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoidcarcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyalinecarcinoma, hypemephroid carcinoma, infantile embryonal carcinoma,carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma,Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma,lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma,medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinouscarcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoidcarcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma,carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportalcarcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceouscarcinoma, renal cell carcinoma of kidney, reserve cell carcinoma,carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma,carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex,small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma,spindle cell carcinoma, carcinoma spongiosum, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, and carcinoma villosum.

The term “carcinoma” also encompasses adenocarcinomas. Adenocarcinomasare carcinomas that originate in cells that make organs which haveglandular (secretory) properties or that originate in cells that linehollow viscera, such as the gastrointestinal tract or bronchialepithelia. Examples include, but are not limited to, adenocarcinomas ofthe breast, lung, pancreas and prostate.

Additional cancers encompassed by the present invention include, forexample, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, small-cell lung tumours,primary brain tumours, malignant pancreatic insulinoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, gliomas,testicular cancer, thyroid cancer, esophageal cancer, genitourinarytract cancer, malignant hypercalcemia, endometrial cancer, adrenalcortical cancer, mesothelioma and medulloblastoma.

In one embodiment, the antisense oligonucleotides are used in thetreatment of a solid tumour. In another embodiment, the antisenseoligonucleotides are used to treat lung cancer, breast cancer, ovariancancer, head and neck cancer or prostate cancer. In another embodiment,the antisense oligonucleotides are used to treat non-small cell lungcancer, breast cancer, ovarian cancer, head and neck cancer or prostatecancer. In another embodiment, the antisense oligonucleotides are usedto treat breast cancer, ovarian cancer, prostate cancer, or non-smallcell lung cancer. In another embodiment, the antisense oligonucleotidesare used to treat colorectal cancer.

In accordance with one embodiment of the present invention, theantisense oligonucleotides are used to inhibit expression of one or moreDNA repair pathway protein(s) in a patient thereby allowing the patientto obtain greater benefit from treatment with a DNA damaging agentand/or an inhibitor of DNA repair or synthesis.

In accordance with one embodiment of the present invention, theantisense oligonucleotides are used to inhibit expression of two or moreDNA repair pathway protein(s) in a patient thereby mimicking a“synthetic lethal” situation.

In another embodiment, the antisense oligonucleotides are used in apatient that already has a known defect in a DNA repair pathway in orderto inhibit expression of a compensatory DNA repair protein therebymimicking or creating a “synthetic lethal” situation.

In another embodiment, the antisense oligonucleotides are used toinhibit a DNA repair pathway and thereby lower the efficiency of ds-DNArepair.

As noted above, the methods provided by the present invention arebroadly applicable to cancer and are not limited to the treatment ofcancers having a defect in a DNA repair mechanism. In one embodiment,however, the invention provides for the use of the antisenseoligonucleotides in the treatment of cancers with one or more defectiveDNA repair mechanisms, for example, the antisense oligonucleotides canbe used in the treatment of cancers with a defective base excisionrepair mechanism, with a defective nucleotide excision repair mechanismor with a defective mismatch repair mechanism. In another embodiment,the invention provides for the use of the antisense oligonucleotides inthe treatment of cancers with one or more defective DNA repairmechanisms wherein the defect is either not well defined or understood.

The antisense oligonucleotides are administered to a subject in anamount effective to achieve the intended purpose. The exact dosage to beadministered can be readily determined by the medical practitioner, inlight of factors related to the patient requiring treatment. Factorswhich may be taken into account when determining an appropriate dosageinclude the severity of the disease state, general health of thesubject, age, weight, and gender of the subject, diet, time andfrequency of administration, the particular components of thecombination, reaction sensitivities, and tolerance/response to therapy.

Antisense oligonucleotides are typically administered parenterally, forexample, by intravenous infusion. Other methods of administeringantisense oligonucleotides are known in the art.

Combination Therapies

In one embodiment, the present invention provides for the use of theantisense oligonucleotides in the treatment of cancer in combinationwith other cancer therapies, such as radiation therapy or chemotherapy.One embodiment of the present invention provides for the use of theantisense oligonucleotides in combination with a cancer therapy thatdamages DNA and/or inhibits DNA repair or synthesis. Suitable cancertherapies include established cancer therapies, as well as novel agentsthat are in clinical trials.

Such combinations may be more effective than either therapy when usedalone. Another embodiment of the invention, therefore, provides for theuse of the antisense oligonucleotides in combination with a cancertherapy that damages DNA and/or inhibits DNA repair or synthesis,wherein the effect of the combination is more than additive orsynergistic.

One embodiment of the invention provides for the use of one or more ofthe antisense oligonucleotides together with a cancer therapy thatdamages DNA and/or inhibits DNA repair or synthesis, wherein the cancertherapy is a platinum drug, inhibitor of PARP, alkylating agent,radiation therapy, or inhibitor of thymidylate synthase. The presentinvention also contemplates the use of the antisense oligonucleotideswith other potential DNA-damaging agents, including, but not limited to,inhibitors of topoisomerases, polymerases, telomerases, helicases,aurora kinase, DNA-dependent kinases, cyclin-dependent kinases, andligases.

In one embodiment, the antisense oligonucleotides are used in thetreatment of cancer in combination with one or more platinum drugs.Non-limiting examples of suitable platinum drugs include cisplatin,carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin, picoplatinand tetranitrate.

In one embodiment, the antisense oligonucleotides are used in thetreatment of cancer in combination with one or more PARP inhibitors.Suitable non-limiting examples of PARP inhibitors include olaparib(AstraZeneca,(4-[(3-{[4-cyclopropylcarbonyl)piperazin-1-yl]carbonyl}-4-fluorophenyl)methyl]phthalazin-1(2H)-one;also known as AZD2281)) or BSI-201 (BiPAR-Sanofi). Other suitableexamples of PARP inhibitors include those described in U.S. PatentPublication Nos. 2005/0227919 or 2009/0098084.

In one embodiment, the antisense oligonucleotides are used in thetreatment of cancer in combination with one or more alkylating agents.Suitable alkylating agents include, for example, melphalan,cyclophosphamide, mechlorethamine or mustinc (HN2), uramustine or uracilmustard, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin,busulfan, and temozolamide.

In one embodiment, the antisense oligonucleotides are used in thetreatment of cancer in combination with radiation therapy. Suitableexamples of radiation therapy include external beam radiotherapy (EBRTor XRT) or teletherapy, brachytherapy or sealed source radiotherapy, orsystemic radioisotope therapy or unsealed source radiotherapy.

In one embodiment, the antisense oligonucleotides are used in thetreatment of cancer in combination with one or more inhibitors ofthymidylate synthase (TS). Suitable inhibitors include, but are notlimited to, the fluoropyrimidine drugs 5-FU, 5-FUdR, capecitabine (anoral form of a pro-drug of 5-FU) and a topical 5-FU cream (Effudex®), aswell as the non-fluoropyrimidine drugs raltitrexed, methotrexate,pemetrexed (Alimta®) and antisense oligonucleotides targeted to the TSgene or mRNA. In a specific embodiment, the antisense oligonucleotidesare used in the treatment of cancer in combination with an antisenseoligonucleotide targeted to the TS gene or mRNA. Suitable anti-TSoligonucleotides include those described in U.S. Patent ApplicationPublication No. 2008/0255066. In one embodiment, the antisenseoligonucleotides are used in the treatment of cancer in combination withan antisense oligonucleotides targeted to the TS mRNA, wherein theanti-TS antisense oligonucleotide comprises the sequence:

(SEQ ID NO: 16) 5′-GCCAGTGGCAACATCCTTAA-3′

In one embodiment, an anti-TS antisense and an anti-BRCA2 antisense areused in combination with a platinum-based chemotherapeutic and aninhibitor of thymidylate synthase including a fluoropyrimidine such as5FU. In specific embodiments, the thymidylate synthase inhibitor ispemetrexed.

In specific embodiments, the anti-BRCA2 antisense is BR1 antisense. Inother specific embodiments, the anti-BRCA2 antisense is BR2 antisense orBR3 antisense.

Clinical Trials in Cancer Patients

One skilled in the art will appreciate that, following the demonstratedeffectiveness of the antisense oligonucleotides in vitro and in animalmodels, they should be tested in Clinical Trials in order to furtherevaluate their efficacy in the treatment of cancer and to obtainregulatory approval for therapeutic use. As is known in the art,clinical trials progress through phases of testing, which are identifiedas Phases I, II, III, and IV. Representative examples of Phase I/IIClinical Trials are provided in the Examples herein.

Initially the antisense oligonucleotides will be evaluated in a Phase Itrial. Typically Phase I trials are used to determine the best mode ofadministration (for example, by pill or by injection), the frequency ofadministration, and the toxicity for the compounds. Phase I studiesfrequently include laboratory tests, such as blood tests and biopsies,to evaluate the effects of a compound in the body of the patient. For aPhase I trial, a small group of cancer patients is treated with aspecific dose of the antisense oligonucleotide. During the trial, thedose is typically increased group by group in order to determine themaximum tolerated dose (MTD) and the dose-limiting toxicities (DLT)associated with the antisense oligonucleotide. This process determinesan appropriate dose to use in a subsequent Phase II trial.

A Phase II trial can be conducted to evaluate further the effectivenessand safety of the antisense oligonucleotides. In Phase II trials, theantisense oligonucleotide is administered to groups of patients witheither one specific type of cancer or with related cancers, using themaximum dosage found to be safe and effective in Phase I trials.

Phase III trials focus on determining how a compound compares to thestandard, or most widely accepted, treatment. In Phase III trials,patients are randomly assigned to one of two or more “arms”. In a trialwith two arms, for example, one arm will receive the standard treatment(control group) and the other arm will receive treatment with theantisense oligonucleotide (investigational group).

Phase IV trials are used to further evaluate the long-term safety andeffectiveness of a antisense oligonucleotide. Phase IV trials are lesscommon than Phase I, II and III trials and will take place after theantisense oligonucleotide has been approved for standard use.

Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example,age, sex, type of cancer) to specific (for example, type and number ofprior treatments, tumour characteristics, blood cell counts, organfunction). Eligibility criteria may also vary with trial phase. Forexample, in Phase I and II trials, the criteria often exclude patientswho may be at risk from the investigational treatment because ofabnormal organ function or other factors. In Phase II and III trialsadditional criteria are often included regarding disease type and stage,and number and type of prior treatments.

Phase I cancer trials usually comprise 15 to 30 participants for whomother treatment options have not been effective. Phase II trialstypically comprise up to 100 participants who have already receivedchemotherapy, surgery, or radiation treatment, but for whom thetreatment has not been effective. Participation in Phase II trials isoften restricted based on the previous treatment received. For trialsthat are investigating the use of the antisense oligonucleotides of theinvention as a first line therapy, for example, the patients selectedfor participation should not have undergone any prior systemic therapy.Phase III trials usually comprise hundreds to thousands of participants.This large number of participants is necessary in order to determinewhether there are true differences between the effectiveness of theantisense oligonucleotide of the present invention and the standardtreatment. Phase III may comprise patients ranging from those newlydiagnosed with cancer to those with extensive disease in order to coverthe disease continuum.

One skilled in the art will appreciate that clinical trials should bedesigned to be as inclusive as possible without making the studypopulation too diverse to determine whether the treatment might be aseffective on a more narrowly defined population. The more diverse thepopulation included in the trial, the more applicable the results couldbe to the general population, particularly in Phase III trials.Selection of appropriate participants in each phase of clinical trial isconsidered to be within the ordinary skills of a worker in the art.

Assessment of Patients Prior to Treatment

Prior to commencement of the study, several measures known in the artcan be used to first classify the patients. Patients can first beassessed, for example, using the Eastern Cooperative Oncology Group(ECOG) Performance Status (PS) scale. ECOG PS is a widely acceptedstandard for the assessment of the progression of a patient's disease asmeasured by functional impairment in the patient, with ECOG PS 0indicating no functional impairment, ECOG PS 1 and 2 indicating that thepatients have progressively greater functional impairment but are stillambulatory and ECOG PS 3 and 4 indicating progressive disablement andlack of mobility.

Patients' overall quality of life can be assessed, for example, usingthe McGill Quality of Life Questionnaire (MQOL) (Cohen et al (1995)Palliative Medicine 9: 207-219). The MQOL measures physical symptoms;physical, psychological and existential well-being; support; and overallquality of life. To assess symptoms such as nausea, mood, appetite,insomnia, mobility and fatigue the Symptom Distress Scale (SDS)developed by McCorkle and Young ((1978) Cancer Nursing 1: 373-378) canbe used.

Patients can also be classified according to the type and/or stage oftheir disease and/or by tumour size.

Administration of the Antisense Oligonucleotides in Clinical Trials

The antisense oligonucleotide is typically administered to the trialparticipants parenterally. In one embodiment, the antisenseoligonucleotide is administered by intravenous infusion. Methods ofadministering drugs by intravenous infusion are known in the art.Usually intravenous infusion takes place over a certain time period, forexample, over the course of 60 minutes.

Monitoring of Patient Outcome

The endpoint of a clinical trial is a measurable outcome that indicatesthe effectiveness of a treatment under evaluation. The endpoint isestablished prior to the commencement of the trial and will varydepending on the type and phase of the clinical trial. Examples ofendpoints include, for example, tumour response rate—the proportion oftrial participants whose tumour was reduced in size by a specificamount, usually described as a percentage; disease-free survival—theamount of time a participant survives without cancer occurring orrecurring, usually measured in months; overall survival—the amount oftime a participant lives, typically measured from the beginning of theclinical trial until the time of death. For advanced and/or metastaticcancers, disease stabilization—the proportion of trial participantswhose disease has stabilised, for example, whose tumour(s) has ceased togrow and/or metastasize (“progress”), can be used as an endpoint. Otherendpoints include toxicity and quality of life.

Tumour response rate is a typical endpoint in Phase II trials. However,even if a treatment reduces the size of a participant's tumour andlengthens the period of disease-free survival, it may not lengthenoverall survival. In such a case, side effects and failure to extendoverall survival might outweigh the benefit of longer disease-freesurvival. Alternatively, the participant's improved quality of lifeduring the tumour-free interval might outweigh other factors. Thus,because tumour response rates are often temporary and may not translateinto long-term survival benefits for the participant, response rate is areasonable measure of a treatment's effectiveness in a Phase II trial,whereas participant survival and quality of life are typically used asendpoints in a Phase III trial.

Pharmaceutical Kits

The present invention additionally provides for therapeutic kitscontaining the antisense oligonucleotide(s) for use in the treatment ofcancer. Individual components of the kit would be packaged in separatecontainers and, associated with such containers, can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. In this case the container means may itself bean inhalant, syringe, pipette, eye dropper, or other such likeapparatus, from which the composition may be administered to a patient.

The components of the kit may also be provided in dried or lyophilisedform and the kit can additionally contain a suitable solvent forreconstitution of the lyophilised components. Irrespective of the numberor type of containers, the kits of the invention also may comprise aninstrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, syringe, pipette,forceps, measured spoon, eye dropper or any such medically approveddelivery vehicle.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It will be understood that theseexamples are intended to describe illustrative embodiments of theinvention and are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 Design of Antisense Oligonucleotides to BRCA2

Three antisense oligonucleotides (OLIGOs) to BRCA2 were designed. Thefirst OLIGO, named BR1, was based on the sequence of a siRNA (siRNAJ-003462-08-0005) commercially available from Dharmacon Inc. (Lafayette,Colo.). BR1 has the following sequence:

[SEQ ID NO: 17] 5′-guaucuCTTGACGTuccuua-3′(40% GC content)

Wherein the lower case letters represent 2′O-methyl RNA and the uppercase letters represent DNA. The OLIGO was fully phosphorothioated. TheBR1 OLIGO targets the coding region, bases 7241-7259 of the BRCA2 mRNA,specifically, the following BRCA2 mRNA sequence:

[SEQ ID NO: 18] 5′-UAAGGAACGUCAAGAGAUAC-3′

Two other OLIGOs, BR2 and BR3, were designed using the NCI web-basedBLAST program. The program was asked to design sequence-specific PCRprimers. Pairs of primers were obtained for the coding region and the3′-UTR of the BRCA mRNA. Based on these sequences, antisense sequenceswere designed and their specificity to BRCA2 mRNA was confirmed usingthe BLAST program.

OLIGO BR2 targets the coding region, bases 8574-8593 of the BRCA2 mRNAsequence, specifically:

[SEQ ID NO: 19] 5′-ACUCGGCCUGCUCGCUGGUA-3′

OLIGO BR2 has the following sequence:

[SEQ ID NO: 20] 5′-uaccagCGAGCAGGccgagu-3′

Wherein the lower case letters represent 2′O-methyl RNA and the uppercase letters represent DNA. The OLIGO was fully phosphorothioated.

OLIGO BR3 targets the 3′-UTR, bases 10615-10634 (131 bases downstream ofthe translation stop site), of the BRCA2 mRNA sequence, specifically:

[SEQ ID NO: 21] 5′-CAGCGUUUGUGUAUCGGGCA-3′

OLIGO BR3 has the following sequence:

[SEQ ID NO: 22] 5′-ugcccgATACACAAacgcug-3′

Wherein the lower case letters represent 2′O-methyl RNA and the uppercase letters represent DNA. The OLIGO was fully phosphorothioated.

Example 2 Inhibition of Proliferation of A549B Cells by an AntisenseOligonucleotide to BRCA2

The effect of an antisense oligonucleotide against BRCA2 onproliferation of non-small cell lung cancer (NSCLC) cells was tested.The BRCA2 antisense oligonucleotide tested in this experiment was BR1,described below. The experiment was carried out as follows.

Cell Culture Techniques.

Cell culture medium was purchased from Wisent, Inc. (St-Bruno, Quebec,Canada). Fetal bovine serum and Lipofectamine 2000 were purchased fromInvitrogen, Inc. Cell culture plasticware was obtained from Invitrogen(Life technologies, Burlington, Ontario, Canada), Fisher Scientific(Unionville, Ontario), and VWR Canlab (Mississauga, Ontario).

Cultured cell lines were maintained in minimum essential medium a withnucleosides plus 10% fetal bovine serum and penicillin (50units/mL)/streptomycin (50 mg/L) (growth medium). Cultures wereincubated in a humidified atmosphere of 5% CO₂ at 37° C. Cultured celllines were maintained and cytotoxicity assays conducted as describedpreviously (3). Rapidly proliferating cells were utilized forestablishing cultures of experimental cells, which were allowed to plateovernight in 25-cm² flasks prior to manipulation. An established cellline of non-small-cell lung carcinoma (NSCLC), A549b, which waspropagated by serial dilution from a single cell of an A549 parent cellline, was used for the establishment of antisense activity ofoligonucleotides. This cell line proliferates with a mean generationtime of approximately 20 hours, and is capable of forming tumours inimmunodeficient mice with a take rate of greater than 90%. Therefore,this cell line is a good model system that can be used in both in vitroand in vivo experiments to test the activity of OLIGOs.

Oligonucleotide Design and Sequences.

Oligonucleotides (OLIGOs) were ordered from Eurogentec (AnaSpec, Inc.,Fremont, Calif., U.S.A.), for which the sequences are synthesized inBelgium. The chemistry of the OLIGOs is such that every phosphodiesterbond in the nucleic acid backbone is a phosphorothioate. Nucleosides onthe outer 6 positions of the sequence contain a methoxy moiety in the2′-position of the ribose. This adds stability to the molecule againstnucleolytic degradation, enhances binding to complementary sequences(decreases AG of binding) and enhances cellular accumulation. The middle8 nucleosides do not contain the methoxy moiety, so as to minimizesteric inhibition of access of ribonuclease H to the double-strandednucleic acid (OLIGO-mRNA hybrid), leading to mRNA degradation.

Cells were treated with either BR1 or control antisense oligonucleotideOLIGO 32. The sequence of OLIGO 32 has no complementary matches with anyknown mRNA sequences. This sequence acts as a control for non-specifictoxicity of the transfection procedure.

[SEQ ID NO: 23] 5′-atgcgcCAACGGTTcctaaa-3′(50% GC content)

The lowercase letters represent 2′-O-methyl RNA and uppercase lettersrepresent DNA.

Transfection of OLIGOs

OLIGOs were introduced into cells with the use of Lipofectamine 2000(LFA2K) (Invitrogen, Burlington, Ontario, Canada). OLIGOs were mixedwith LFA2K at a ratio of 0.2 μg/ml per 10 nM OLIGO. The mixture wasprepared at 11× the final concentration to which cells were exposed, sothat 200 μL was added to 2 mL of medium in which cells were plated. Ascontrols, cells were exposed to medium alone (no treatment), LFA2K aloneat concentrations equivalent to those used in combination with OLIGOs(in some experiments only the maximum LFA2K concentration was used as acontrol), or OLIGO having no complementarity with any human mRNA. TheOLTGO/LFA2K mixtures were then incubated at room temperature for 20minutes, according to instructions supplied with the LFA2K, followed byaddition of the OLIGOs to the cell medium. The OLIGO/LFA2K mix was thenincubated (37° C.) on the cells for 4 hours, after which a second volumeof medium was added. Cells were then incubated for 20 hours. Followingthis incubation, the OLIGO-containing medium was replaced with drug-freemedium (i.e. medium without OLIGOs) and the cells were incubated for anadditional 4 days. Following the 4-day incubation, the proliferation ofthe treated cells (fold-increase in cell number) was calculated as apercentage of that of control cells. Cell numbers were enumerated on anelectronic particle counter (Beckman Coulter, Mississauga, ON). For thepurpose of determining the anti-proliferative effect of antisenseOLIGOs, the proliferation of cells treated with antisense OLIGOs wascalculated as a percent of that of cells treated with an equivalentconcentration of non-complementary control OLIGO (in this case OLIGO 32)mixed with an equivalent amount of LFA2K.

Initial experiments were performed to optimize the ratio of LFA2K to theantisense oligonucleotide, and to determine what concentration of OLIGOwas capable of inhibiting cell proliferation. The results of thisexperiment are shown in FIG. 1. As a single agent, OLIGO BR1, mixed with0.2 μg/mL LFA2K, was able to inhibit proliferation of A549b cells byover 50%, compared with the control OLIGO 32 mixed with 0.2 μg/mL LFA2K.

Example 3 Inhibition of Proliferation of A549B Cells Pretreated With anAntisense Oligonucleotide to BRCA2 by Olaparib

This experiment examined the effect of pre-treating A549b cells with theBRCA2 antisense oligonucleotide BR1 on the ability of the PARP (poly(ADPribose) polymerase) inhibitor olaparib to inhibit proliferation of thesecells. These experiments were carried out as follows.

Cells were cultured and maintained as described in Example 2. Theantisense oligonucleotide sequences used were also as described inExample 2.

Cells were treated with OLIGOs and/or olaparib as follows.

OLIGOs were introduced into cells with the use of Lipofectamine 2000(LFA2K) (Invitrogen, Burlington, Ontario, Canada). OLIGOs were mixedwith LFA2K at a ratio of 0.2 μg/ml per 10 nM OLIGO. The mixture wasprepared at 11× the final concentration to which cells were exposed, sothat 200 μL was added to 2 mL of medium in which cells were plated.After incubating at room temperature for 20 minutes, according tomanufacturer's instructions, the OLIGOs were added to the cell medium.The OLIGO/LFA2K mix was then incubated (37° C.) on the cells for 4hours, after which a second volume of medium was added. Cells were thenincubated for 20 hours. Following this incubation, the OLIGO-containingmedium was replaced with OLIGO-free medium.

For cells treated with the drug olaparib, for the purposes ofdetermining whether inhibitory activity was enhanced by the OLIGOpretreatment, olaparib was added at this time, in concentrations of from0.01 μM to 3 μM. At this point, replicate flasks from theOLIGO-treatment were used to enumerate cell content, as this variedamong treatments over the initial 24-hour exposure. This was done sothat the effect of treatment with olaparib could be ascertained based onthe cell population that was present at the time of initiation ofexposure to olaparib. Exposure to the olaparib was initiated by additionof 0.2-volume of a preparation of the drug, at 6× final concentration ingrowth medium, to the fresh drug-free medium on the cells (1 ml of druginto 5 ml of medium). Following a further 4-day incubation, theproliferation of the treated cells (fold-increase in cell number) wascalculated as a percentage of that of control cells. Cell numbers wereenumerated on an electronic particle counter (Beckman Coulter,Mississauga, ON). As indicated in Example 1, for the purpose ofdetermining the anti-proliferative effect of antisense OLIGOs alone, theproliferation of cells treated with antisense OLIGOs was calculated as apercent of that of cells treated with an equivalent concentration ofnon-complementary control OLIGO (in this case OLIGO 32). However, forthe purpose of determining the anti-proliferative effect of olaparibagainst antisense or non-complementary (control) OLIGO-treated cells,the proliferation was calculated as a percent of cells treated with therespective OLIGO.

The results of this experiment are shown in FIG. 2, which shows thatpretreatment of A549b cells with BR1 enhanced the anti-proliferativeeffect of olaparib by over 40% at a given concentration of olaparib.Interpreted in a different manner, if these curves are conservativelyextrapolated, the concentration of olaparib required to inhibitproliferation of A549b cells by 50% (IC50) appears to be several ordersof magnitude greater in the absence of BR1 than in its presence(approximate IC50 values of 5 and 0.05, respectively). This indicatesthe importance of PARP to the survival of drug-treated cells anddemonstrates that in spite of the redundancies evolved into theDNA-repair system, PARP is essential to the maintenance of DNA integrityin the absence of BRCA2. It also suggests that BRCA1 does not functionas a back-up system in the absence of BRCA2.

Example 4 Effect of an Antisense Oligonucleotide to BRCA2 Alone and inCombination With Olaparib on Proliferation of NSCLC Cells

The experiments described in Examples 2 and 3 were repeated. Theexperimental steps were the same. However, the amount of LFA2K useddiffered in some cases, and additional concentrations of olaparib weretested with cells pretreated with BR1 or control oligonucleotides. Theresults are shown in FIGS. 3 to 6. Modifications with respect to theamount of BR1, LFA2K and the concentrations of olaparib used arc shownon the Figures themselves.

The results showed that OLIGO BR1, when combined with an optimalconcentration of transfection reagent, again inhibited proliferation onits own as shown in FIG. 3, and enhanced the antiproliferative activityof olaparib, as shown in FIG. 4. As shown in FIGS. 5 and 6, a higherconcentration of BR1 had greater inhibition as a single agent, andslightly greater enhancement of olaparib toxicity compared to thepreviously used 10 nM.

Example 5 Effect of Pretreatment of A549B Cells With AntisenseOligonucleotides to BRCA2 on the Ability of Cisplatin to InhibitProliferation

The ability of anti-BRCA2 OLIGOs to enhance the anti-proliferativeactivity of a PARP inhibitor as shown in Examples 3 and 4 suggested thatDNA damage occurs spontaneously in proliferating tumour cells, or thatthese enzymes are also involved in the normal replication of DNA. Assuch, it suggested that if DNA were damaged by addition of achemotherapy drug, inhibition of BRCA2 might also enhance thecytotoxicity of the drug. In order to determine if this was the case,therefore, cells were treated with the DNA cross-linking agent cisplatinfollowing transfection with OLIGO BR1 or with an antisense OLIGO thattargets the 3′-UTR of the BRCA2 mRNA, OLIGO BR3.

These experiments were carried out essentially as described in Example3, with the following changes. The drug tested was cisplatin instead ofolaparib, and cisplatin was tested in concentrations ranging from 0.5 μMto 2 μM.

The BRCA2 antisense oligonucleotides used were BR1 and BR3. In theseexperiments, the control oligonucleotide was OLIGO 491S (also referredto as OLIGO 91S). OLIGO 491S is a control sequence that, like OLIGO 32,has no matching complementary mRNA sequences. The sequence of the OLIGO491S oligonucleotide is:

[SEQ ID NO: 24] 5′-ggagtgCGTGAGTCgatgta-3′(55% GC content)

The results of these experiments are shown in FIGS. 7 and 8. Compared tothe non-complementary control, in this case OLIGO 491S, both BR1 and BR3were able to enhance the cytotoxicity of cisplatin by approximately 5-to 10-fold. To put these results in perspective, if such enhancement ofantitumour activity could be achieved in patients, it wouldsignificantly decrease toxicities related to hearing loss, kidneydamage, nausea and vomiting, and bone marrow depression.

Example 6 Ability of a Combination of Antisense Oligonucleotides toBRCA2 to Inhibit Proliferation of A549B Cells

The following experiment was carried out to test the effect of combiningthe anti BRCA2 OLIGOs, BR1 and BR3, at concentrations that had verylittle inhibitory activity as single agents, on the proliferation ofA549b cells.

The experiments were carried out as described in Example 2, with theexception that the control OLIGO used was OLIGO 491S. The results areshown in FIG. 9 and demonstrate that, at concentrations of BR1 and BR3that had very little inhibitory activity as single agents, these twoOLIGOs inhibited A549b proliferation greater than would be predicted byan additive effect.

Example 7 Effect of Pretreatment With Antisense Oligonucleotides toBRCA2 on the Ability of Cisplatin to Inhibit Proliferation of A549BCells

The following experiment was carried out in order to determine whetherthe combination of BR1 and BR3 tested in Example 6 could also enhancethe effect of cisplatin on the proliferation of A549b cells. Theexperiment was carried out as described in Example 3, except that theconcentration of BR1 and BR3 antisense oligonucleotides were asdescribed in Example 6, the control oligonucleotide used was OLIGO 4915,and the drug used was cisplatin.

The results of this experiment are shown in FIGS. 10 and 11. Whenpretreatment of A549 cells with a combination of OLIGOs BR1 and BR3 wasfollowed by exposure to cisplatin, the anti-proliferative activity ofcisplatin was enhanced approximately 5-fold by concentrations of OLIGOthat had negligible effect on proliferation on their own or even atconcentrations equivalent to that of the combination.

Example 8 Effect of a Combination of an Antisense Oligonucleotide toBRCA2 and an Antisense Oligonucleotide to Thymidylate Synthase on theProliferation of A549B Cells

OLIGO 83 is an antisense oligonucleotide targeted to the 3′-untranslatedregion of mRNA of thymidylate synthase (TS) and down-regulates TS mRNAand protein, inhibits proliferation of cancer cells, and enhancescytotoxicity of TS-inhibitory drugs such as 5-fluorodeoxyuridine andpemetrexed (4, 5). Given that TS-inhibitors are often used incombination with platinum drugs against some tumour types, such ascarcinomas of the breast, lung, colon, and head and neck, thecombination of OLIGOs targeting both TS and BRCA2 was tested todetermine whether they could be used to enhance the antitumour activityof such drug combinations. Initially, the combination of OLIGO 83 andBR3 was tested in A549b cells to determine the effect of thiscombination on cell proliferation.

The experiment was carried out as described in Example 2, except thatthe oligonucleotides tested were BR3 and OLIGO 83. The controloligonucleotide was OLIGO 491S. The sequence of OLIGO 83 is:

[SEQ ID NO: 25] 5′-gccaguGGCAACATccuuaa-3′(50% GC content).

The lowercase letters represent 2′-O-methyl RNA and uppercase lettersrepresent DNA. OLIGO 83 is fully phosphorothioated.

The results of this experiment are shown in FIG. 12. In this preliminaryassay, compared to an equivalent concentration of non-targeting OLIGO(491S), the combination of BR3 and OLIGO 83 caused greater inhibition ofproliferation than would be predicted based on the inhibition caused byeach OLIGO alone, at the respective concentrations. For example,relative proliferation following treatment with 10 nM of BR3 wasapproximately 85% and following treatment with 20 nM OLIGO 83 wasapproximately 20%. The relative proliferation following combinedtreatment was approximately 0%. This result suggests that there may be agreater than additive or synergistic anti-tumour effect of this OLIGOcombination, and that this combination could potentially synergisticallyenhance the effect of drugs such as 5-fluorouracil and cisplatin whenadministered together.

Example 9 Effect of Pretreatment of A549B Cells With Anti-BRCA2 OligoBR1 on Cytotoxicity of Melphalan Against Medium Density A549B Cells

This experiment examined the effect of pretreating A549b cells with theBRCA2 antisense oligonucleotide BR1 on the cyotoxicity of melphalan.

These experiments were carried out essentially as described in Example3, with the following changes. The drug tested was melphalan inconcentrations ranging from 2 μM to 10 μM.

The BRCA2 antisense oligonucleotide used was BR1. In these experiments,the control oligonucleotide was OLIGO 32.

The results of these experiments are shown in FIG. 13. Compared to thenon-complementary control, in this case OLIGO 32, BR1 was able toenhance the cytotoxicity of melphalan.

Example 10 Effect of Pretreatment of A549B Cells With Anti-BRCA2 OLIGOBR1 on Cytotoxicity of Carboplatin Against Medium Density A549B Cells

This experiment examined the effect of pretreating A549b cells with theBRCA2 antisense oligonucleotide BR1 on the cyotoxicity of carboplatinagainst A549b cells.

These experiments were carried out essentially as described in Example3, with the following changes. The drug tested was carboplatin atconcentrations ranging from 5 μM to 40 μM.

The BRCA2 antisense oligonucleotides used were BR1. In theseexperiments, the control oligonucleotide was OLIGO 32.

The results of these experiments are shown in FIG. 14. Compared to thenon-complementary control, in this case OLIGO 32, BR1 was able toenhance the cytotoxicity of carboplatin.

Example 11 Effect of Pretreatment of A549B Cells With Anti-BRCA2 OLIGOBR1 on Cytotoxicity of Oxaliplatin Against Low Density A549B Cells

This experiment examined the effect of pretreating A549b cells with theBRCA2 antisense oligonucleotide BR1 on the cyotoxicity of oxaliplatin.

These experiments were carried out essentially as described in Example3, with the following changes. The drug tested was oxaliplatin wastested in concentrations ranging from 0.211M to 2.5 μM.

The BRCA2 antisense oligonucleotide used was BR1. In these experiments,the control oligonucleotide was OLIGO 32.

The results of these experiments are shown in FIG. 15. Compared to thenon-complementary control, in this case OLIGO 32, BR1 was able toenhance the cytotoxicity of oxaliplatin at lower doses.

Example 12 Antisense TS OLIGO 83 and Antisense BRCA2 OLIGO BR1 ActIndependently to Reduce Thymidylate Synthase and BRCA2 mRNA Levels

This experiment tested the effect of treatment of antisense OLIGOs 83and BR1 on both TS and BRCA2 mRNA levels.

Briefly, A549 cells seeded at a density of 2.0×10⁵ per flask weretransfected with 20 nM of OLIGO specific for each target or controlOLIGO using Lipofectamine 2000 as the transfection reagent. Twenty fourhours post-transfection mRNA was extracted and reverse transcribed intocDNA. RT-qPCR was performed for target mRNA levels using TaqMan reagentsaccording to established protocols.

As shown in FIG. 16, antisense TS (labeled SARI 83) and antisense BR1(labeled T1) OLIGO act independently to reduce TS and T1 mRNA. Inparticular, antisense OLIGO-mediated reduction in TS mRNA has nosignificant effect on BRCA2 mRNA, and antisense OLIGO-mediated reductionin BRCA2 mRNA has no significant effect on TS mRNA. In addition,simultaneous treatment with antisense TS OLIGO and antisense BR1 OLIGOreduces TS and BRCA2 mRNAs to the same degree as independent,non-concomitant treatment. Therefore, additive or greater-than-additiveeffects of antisense TS and BR2 OLIGOs on tumour cell proliferationcannot be attributed to additive/greater-than-additive capacity toreduce mRNA.

Example 13 Synergistic Effect of Antisense TS OLIGO and Antisense BR1

This experiment examined the combined effect of BRCA2 antisenseoligonucleotide BR1 and antisense TS oligonucleotide OLIGO 83 onproliferation of A549b cells.

These experiments were carried out essentially as described in Example2, after which one volume of medium was added, and cells were furtherincubated for 20 hours. OLIGO-containing medium was removed and replacedwith fresh medium. Cells were incubated for 4 days, at which time cellnumber was counted. Proliferation was calculated as a percent ofnon-targeting OLIGO-treated controls.

The experimental steps were the same for the experiments detailed inFIGS. 17A-17E. However, the amount of LFA2K used differed in some cases,and concentrations of OLIGO BR1 and OLIGO 83 were varied. Modificationswith respect to the amount of BR1, OLIG083 and LFA2K are shown on theFigures themselves.

As is shown in FIGS. 17A-17E, the combination of OLIGO BR1 and OLIGO 83caused greater inhibition of proliferation than what would be expectedby an additive effect of each OLIGO alone.

Example 14 Synergistic Effect of Antisense TS OLIGO and Antisense BR3

This experiment examined the combined effect of BRCA2 antisenseoligonucleotide BR3 and antisense TS oligonucleotide OLIGO 83 onproliferation of A549b cells.

These experiments were carried out essentially as described in Example2, after which one volume of medium was added, and cells were furtherincubated for 20 hours. OLIGO-containing medium was removed and replacedwith fresh medium. Cells were incubated for 4 days, at which time cellnumber was counted. Proliferation was calculated as a percent ofnon-targeting OLIGO-treated controls.

The experimental steps were the same for the experiments detailed inFIGS. 18A and 18B. However, the amount of LFA2K used differed in somecases, and concentrations of OLIGO BR3 and OLIGO 83 were varied.Modifications with respect to the amount of BR3, OLIG083 and LFA2K areshown on the Figures themselves.

As is shown in FIGS. 18A and 18B, the combination of OLIGO BR3 andOLIGO83 caused greater inhibition of proliferation than what would beexpected by an additive effect of each OLIGO alone.

Example 15 Effect of Combination of Four Anti-BRCA2 SiRNAs onCytotoxicity of Cisplatin in A549B Cells

This experiment examined the effect of four anti-BRAC2 siRNA, total 5nM, on the cytotoxity of cisplatin in A549b cells.

These experiments were carried out essentially as described in Example2, after which one volume of medium was added, and cells were furtherincubated for 20 hours. siRNA-containing medium was removed and replacedwith fresh medium. Cisplatin was added to the final concentrationindicated, and as described in Example 3. Cells were incubated for 4days, at which time cell number was counted. Proliferation wascalculated as a percent of siRNA-treated, non-cisplatin-treatedcontrols.

The results of this experiment are shown in FIG. 19, which shows thatpretreatment of A549b cells with four anti-BRCA2 siRNAs enhanced theanti-proliferative effect of cisplatin.

Example 16 Effect of Anti-RAD51 SiRNA on Proliferation of PANC-1Pancreatic Carcinoma Cells

This experiment examined the effect of four different siRNA moleculesagainst RAD51 on proliferation of PANC-1 pancreatic carcinoma cells.

These experiments were carried out essentially as described in Example2, after which one volume of medium was added, and cells were furtherincubated for 20 hours. siRNA-containing medium was removed and replacedwith fresh medium. Cells were incubated for 4 days, at which time cellnumber was counted. Proliferation was calculated as a percent ofcontrols.

The results of this experiment are shown in FIG. 20, which shows thatfour different siRNA molecules against RAD51 inhibited proliferation ofPANC-1 pancreatic carcinoma cells by 40 to 50% at 5 nM.

Example 17 Effect of Anti-RAD51 SiRNA on Proliferation of A549B Cells

This experiment examined the effect of siRNA RADb molecules againstRAD51 on proliferation of A549b cells.

These experiments were carried out essentially as described in Example2, after which one volume of medium was added, and cells were furtherincubated for 20 hours. siRNA-containing medium was removed and replacedwith fresh medium. Cells were incubated for 4 days, at which time cellnumber was counted. Proliferation was calculated as a percent ofcontrols.

The results of this experiment are shown in FIG. 21, which shows thatsiRNA RADb against RAD51 inhibited proliferation of A549b cells by over50% at 2 nM.

Example 18 Effect of Combined TS SiRNA and BRCA2 SiRNA on A549B CellSensitivity to Treatment With Cisplatin and 5FUDR

This experiment examined the effect of combined TS siRNA and BRCA2 siRNAon A549b sensitivity to cisplatin and 5FUdR.

A549 cells were transfected with control non-targeting siRNA (20 nM) orTS siRNA (10 nM) and BRCA2 siRNA (10 nM). Cisplatin (4 μM) and 5FUdR (10nM) or vehicle control was added 24 hours later. Cells were allowed toproliferate for 96 hours and then counted (Coulter counter). Cisplatinand 5FUdR treatment, or TS siRNA plus BRCA2 siRNA treatment, reducedproliferation. As shown in FIG. 22 combined treatment with both siRNAsand both drugs reduced proliferation further than treatment with siRNAsor drugs alone.

REFERENCES

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Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention. All such modifications as would be apparent to oneskilled in the art are intended to be included within the scope of thefollowing claims.

TABLE 3 Sequences SEQ ID NO: SEQUENCES: NOTES:  15′-guaucuCTTGACGTuccuua-3′ BR1  gapmer-lowercase  letters represent 2′-O-methyl RNA; phosphorothioate throughout the  entire lengthof the OLIGO  2 5′-uaccagCGAGCAGGccgagu-3′ BR2  gapmer-lowercase letters represent  2′-O-methyl RNA; phosphorothioate throughout theentire length of  the OLIGO  3 5′-ugcccgATACACAAacgcug-3′ BR3 gapmer-lowercase  letters represent  2′-O-methyl RNA; phosphorothioatethroughout the  entire length  of the OLIGO  45′-CAGCGTTTGTGTATCGGGCA-3′ BRCA2 Antisense  5 5′-TTGGATCCAATAGGCAT-3′BRCA2 Antisense  6 5′-TACGTACTCCAGAACATTTAA-3′ BRCA2 Antisense  75′-TTGGAGGAATATCGTAGGTAA-3′ BRCA2 Antisense  85′-CAGGACACAATTACAACTAAA-3′ BRCA2 Antisense  9 5′-UAAAUAGCAAGUCCGUUUC-3′BRCA2 siRNA “A” 10 5′-UAAUGAAGCAUCUGAUACC-3′ BRCA2 siRNA “B” 115′-UAUUAAACCUGCAUUCUUC-3′ BRCA2 siRNA “C” 12 5′-GUAUCUCUUGACGUUCCUUA-3′BRCA2 siRNA “D” 13 5′-GTATCTCTTGACGTTCCTTA-3′ BR1 DNA 145′-TACCAGCGAGCAGGCCGAGT-3′ BR2 DNA 15 5′-TGCCCGATACACAAACGCTG-3′ BR3 DNA16 5′-GCCAGTGGCAACATCCTTAA-3′ OLIGO83 17 5′-guaucuCTTGACGTuccuua-3′BR1 modified  (lowercase  letters represent  2′-O-methyl RNA) 185′-UAAGGAACGUCAAGAGAUAC-3′ BR1 target 19 5′-ACUCGGCCUGCUCGCUGGUA-3′BR2 target 20 5′-uaccagCGAGCAGGccgagu-3′ BR2 modified  (lowercase letters  represent 2′-O-methyl RNA) 21 5′-CAGCGUUUGUGUAUCGGGCA-3′BR3 target 22 5′-ugcccgATACACAAacgcug-3′ BR3 modified  (lowercase letters represent  2′-O-methyl RNA) 23 5′-augcgcCAACGGTTccuaaa-3′OLIGO32  (gapmer control) 24 5′-ggagugCGTGAGTCgaugua-3′ OLIGO491S (gapmer control) 25 5′-gccaguGGCAACATccuuaa-3′ OLIGO83  (lowercase letters  represent  2′-O-methyl RNA) 26 5′-CUGCAUCUGCAUUGCCAUUA-3′prior art RAD51  target 27 5′-GGCUUCACUAAUUCC-3′ prior art RAD51  target28 5′-GUAAUGGCAAUGCAGAUGC-3′ prior art RAD51  target 295′-GAAUGGGUCUGCACAGAUUC-3′ RAD51 target 30 5′-gaaucuGTGCAGACccauuc-3′RAD51 antisense  gapmer (lowercase letters represent  2′-O-methyl RNA)31 5′-GCAAGCCAGCTGAGGGCACA-3′ DNA-PK antisense 325′-GGGCATTCCAAGGCTTCCCCA-3′ DNA-PK antisense 335′-GGGCTCCCATCCTTCCCAGG-3′ DNA-PK antisense 345′-AGGGGCCTTCTCATGACCCAGG-3′ DNA-PK antisense 355′-ACTGCTGGATTGGCACCTGCT-3′ DNA-PK antisense 365′-TGGGGTCTGTTGCCTGGTCC-3′ DNA-PK antisense 37 5′-AAUUUCUUCACAUCGUUGG-3′siRNA against  RAD51 “A” 38 5′-UUAUCCAGGACAUCACUGC-3′ siRNA against RAD51 “B” 39 5′-UGAGCUACCACCUGAUUAG-3′ siRNA against  RAD51 “C” 405′-UGAUGCAUGGGCGAUGAUA-3′ siRNA against  RAD51 “D” 415′-GUAUCUCUUGACGUUCCUUA-3′ BR1 RNA 42 5′-UACCAGCGAGCAGGCCGAGU-3′ BR2 RNA43 5′-UGCCCGAUACACAAACGCUG-3′ BR3 RNA 44 5′-GAATCTGTGCAGACCCATTC-3′RAD51 antisense 45 5′-gaaucuGTGCAGACccauuc-3′ RAD51  gapmer-lowercase  letters represent   2′-O-methyl RNA; phosphorothioate throughout the entire length of  the OLIGO

1-47. (canceled)
 48. A method of treating cancer in a subject comprisingadministering to the subject an effective amount of an antisenseoligonucleotide comprising a sequence complementary to an mRNA encodinga DNA double strand break repair protein, wherein the antisenseoligonucleotide has a length between 7 and 100 nucleotides in length,between about 12 and about 50 nucleotides, between about 12 and 35nucleotides or between about 12 and 30 nucleotides.
 49. The methodaccording to claim 48, wherein the mRNA encodes BRCA2 or RAD51.
 50. Themethod according to claim 48, wherein the antisense oligonucleotidecomprises (a) at least 7 consecutive nucleotides of the sequence as setforth in any one of SEQ ID NOs: 1, 2, 3, 13, 14 or 15; (b) at least 10consecutive nucleotides of the sequence as set forth in any one of SEQID NOs: 1, 2, 3, 13, 14 or 15; or (c) the sequence as set forth in anyone of SEQ ID NOs: 1, 2, 3, 13, 14 or
 15. 51. The method according toclaim 48, wherein the antisense oligonucleotide comprises (a) one ormore phosphorothioate bonds; (b) one or more 2′-O-methyl modifiednucleotides; (c) one or more 2′-O-methoxyethyl (2′-MOE) modifiednucleotides; or (d) both RNA and DNA nucleotides.
 52. The methodaccording to claim 48, wherein the antisense oligonucleotide is a gapmerantisense oligonucleotide.
 53. The method according to claim 48, whereinthe cancer is a solid tumour.
 54. The method according to claim 53,wherein the cancer is lung cancer, colorectal cancer, gastric cancer,esophageal cancer, breast cancer, ovarian cancer, head and neck canceror prostate cancer.
 55. The method according to claim 48, wherein theantisense oligonucleotide is administered in combination with a secondantisense oligonucleotide of between 7 and 100 nucleotides in lengthcomprising a sequence complementary to an mRNA encoding a DNA doublestrand break repair protein.
 56. The method according to claim 55,wherein (a) each antisense oligonucleotide comprises a sequencecomplementary to a mRNA encoding BRCA2; (b) the first antisenseoligonucleotide comprises a sequence complementary to a mRNA encodingBRCA2 and the second antisense oligonucleotide comprises a sequencecomplementary to an mRNA encoding a different DNA double strand breakrepair protein in the homologous recombination repair pathway; or (c)the first antisense oligonucleotide comprises a sequence complementaryto a mRNA encoding BRCA2 and the second antisense oligonucleotidecomprises a sequence complementary to a mRNA encoding a DNA doublestrand break repair protein in the non-homologous end joining repairpathway.
 57. The method according to claim 48, wherein the antisenseoligonucleotide is administered in combination with another cancertherapy.
 58. The method according to claim 57, wherein the cancertherapy results in DNA damage, inhibition of a DNA repair pathway orinhibition of DNA synthesis.
 59. The method according to claim 57,wherein the cancer therapy comprises (a) radiation therapy, treatmentwith a chemotherapeutic drug and/or treatment with an antisenseoligonucleotide; (b) treatment with an alkylating agent; (c) treatmentwith a platinum-based chemotherapeutic; (d) radiation therapy; (e)treatment with a PARP inhibitor; (f) treatment with an inhibitor ofthymidylate synthase; wherein optionally the inhibitor of thymidylatesynthase is (a) an antisense oligonucleotide targeted to thymidylatesynthase mRNA; or (b) a chemotherapeutic drug, wherein optionally thechemotherapeutic drug is 5-FU, 5-FUdR, capecitabine, raltitrexed,methotrexate or pemetrexed.
 60. An antisense oligonucleotide of between7 and 100 nucleotides in length comprising at least 7 consecutivenucleotides of the sequence as set forth in any one of SEQ ID NOs: 2, 3,14, 15, 30, 31, 32, 33, 34, 35 or
 36. 61. The antisense oligonucleotideaccording to claim 60, wherein the antisense oligonucleotide comprisesthe sequence as set forth in any one of SEQ ID NOs: 2, 3, 14, 15, 30,31, 32, 33, 34, 35 or
 36. 62. The antisense oligonucleotide according toclaim 60, comprising (a) one or more phosphorothioate bonds; (b) one ormore 2′-O-methyl modified nucleotides; (c) one or more 2′-O-methoxyethyl(2′-MOE) modified nucleotides; or (d) both RNA and DNA nucleotides. 63.The antisense oligonucleotides according to claim 60, wherein theantisense oligonucleotide is a gapmer antisense oligonucleotide.
 64. Apharmaceutical composition comprising one or more of the antisenseoligonucleotides according to claim 60.